Claudia Escher1, Reto Ossola1, Patrina Gunness2

Proteomic Analysis of Acetaminophen Toxicity in 3D Human Liver Microtissues
Claudia Escher , Reto Ossola , Patrina Gunness , Magdalena Bober , Fiona Grimm , Lukas Reiter , Yulia Butscheid , Simon Messner
1
Biognosys AG, Schlieren, Switzerland, www.biognosys.ch
2
InSphero AG, Schlieren, Switzerland, www.insphero.com
1
1
2
1
1
1
1
2
1890-262
Data-independent acquisition
-
Protein ID
1
Protein name
Peptide
20
40
60
80
100
Retention time [min]
120
Upregulated
Oxidation reduction (1)
Drug metabolic process (2)
Mitochondrial transport (3)
Xenobiotic metabolic process (5)
Downregulated
Response to calcium ion (1)
Response to inorganic substance (2)
Response to metal ion (3)
Fatty acid metabolic process (4)
Leukotriene metabolic process (6) / Icosanoid metabolic process (16)
Response to wounding (14) / Acute inflammatory response (22)
Replicate 1
Replicate 2
Mass spectrometric raw data show a
drastic induction of CYP1A2 already
at subtoxic concentrations of acetaminophen. No effect on cell viability is detectable at these levels.
Donor I
0.9
Donor II
0.8
0.7
0.6
10
100
1000
10000
Acetaminophen concentration [uM]
1

3
2.5
2
Donor I
Donor II
1
0.5
10
100
1000
10000
Acetaminophen concentration [uM]
1.8
1.6
1.4
Donor I
1.2
Donor II
1
0.8
1
10
100
1000
10000
Acetaminophen concentration [uM]
1.4
1.3
1.2
1.1
1
Donor I
0.9
Donor II
0.8
0.7
0.6
1
4
3.5
3
Donor I
2
Donor II
1.5
1
0.5
1
10
100
1000
10000
Acetaminophen concentration [uM]
10
100
1000
10000
Acetaminophen concentration [uM]
Cytochrome P450 1A2 (UniProt P05177)
4.5
2.5
10
100
1000
10000
Acetaminophen concentration [uM]
Bile salt sulfotransferase (UniProt Q06520)
CYP3A5 (UniProt P20815)
CYP2E1 (UniProt P05181)
1.5
Fold-change vs. Control Sample
Fold-change vs. Control Sample
Fold-change vs. Control Sample
1
Gluronoyltransferases are upregulated in 3D Liver Microtissues
from both donors with increasing acetaminophen concentration
(above). The bile salt sulfotransferase (right) shows a donor II-specific upregulation.
In contrast, a donor-specific upregulation is seen for cytochrome
P450 2E1 in donor I (below). These observations might explain the
higher acetaminophen toxicity observed in donor I as more of the
toxic NAPQI metabolite might be produced.
1
Replicate 3
1.1
Fold-change vs. Control Sample
1103.02 3309.07
10
56
1.2
Fold-change vs. Control Sample
367.67
3
Unbiased global discovery approach reveals early upregulation of CYP1A2 protein
O75608
Acyl-protein thioesterase 1
TYEGMMHSSCQQEMMDVK
731.29103
Enoyl-CoA hydratase domain-containing protein 2, mitochondrial
LSGTEAHVLGLVNHAVAQNEEGDAAYQR
738.11669
P30711
Glutathione S-transferase theta-1
VEAAVGEDLFQEAHEVILK
699.70027
P55157
Microsomal triglyceride transfer protein large subunit
EFYSYQNEAVAIENIKR
692.01285
Q99733
Nucleosome assembly protein 1-like 4
LDNVPHTPSSYIETLPK
637.66679
P53621
Coatomer subunit alpha
GVNWAAFHPTMPLIVSGADDR
752.04063
Q9UL12
Sarcosine dehydrogenase, mitochondrial
HWHADLRPDDSPLEAGLAFTCK
634.8064
P21397
Amine oxidase [flavin-containing] A
EIPTDAPWEAQHADKWDK
713.00435
Q15149
Plectin
DALDGPAAEAEPEHSFDGLR
699.6555
Q00796
Sorbitol dehydrogenase
MHSVGICGSDVHYWEYGR
718.31577
P80404
4-aminobutyrate aminotransferase, mitochondrial
MLDLYSQISSVPIGYSHPALLK
811.43465
P30153
Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit
IGPILDNSTLQSEVKPILEK
732.08249
P23378
Glycine dehydrogenase [decarboxylating], mitochondrial
IQPVEVDKYGNIDAVHLK
510.27965
P19338
Nucleolin
VEGTEPTTAFNLFVGNLNFNK
1156.5815
P30048
Thioredoxin-dependent peroxide reductase, mitochondrial
AFQYVETHGEVCPANWTPDSPTIKPSPAASK
847.1594
Q9NQ94
APOBEC1 complementation factor
NNWGQPVYQLHSAIGQDQR
737.69451
Q13907
Isopentenyl-diphosphate Delta-isomerase 1
ITFPGCFTNTCCSHPLSNPAELEESDALGVR
1160.527
P05089
Arginase-1
RPIHLSFDVDGLDPSFTPATGTPVVGGLTYR
822.17802
P00439
Phenylalanine-4-hydroxylase
THACYEYNHIFPLLEK
678.99625
Q9H3P7
Golgi resident protein GCP60
GPGASGEQPEPGEAAAGGAAEEAR
1083.4883
P00966
Argininosuccinate synthase
FAELVYTGFWHSPECEFVR
792.03689
2
Retrospective data analysis
Upregulated
Protein
Cytochrome P450 1A2
Sulfiredoxin-1
Cytochrome P450 2C8
Cytochrome P450 4F3
0
122.56
3
Donor II
1.3
2
14
12
10
8
6
Donor I
4
Donor II
2
0
1
10
100
1000
10000
Acetaminophen concentration [uM]
Mass/Charge
Q86YB7
Data-independent acquisition
40.85
1
Donor I
1.4
Proteins with highest regulation in liver microtissues upon acetaminophen treatment
Peptide spectral library
Relative intensity
HRM-MS™ technology
HRM-MS is a novel mass spectrometric approach for global proteome profiling and biomarker discovery. Data-independent
acquisition (DIA) allows the measurement of all detectable peptide signals in a given sample e.g. over a treatment range. Subsequent matching with a previously generated peptide spectral library enables reproducible and accurate quantification
of thousands of proteins in a single instrument measurement.
Thus, HRM-MS™ combines the scale of shotgun mass spectrometry with the analytical accuracy of targeted proteomics especially when protein quantification across multiple samples is
required.
13.62
2
UDP-glucuronosyltransferase 2B15 (UniProt
P54855)
UDP-glucuronosyltransferase 1-6 (UniProt
P19224)
Fold-change vs. Control Sample
Donor-specific sensitivity to acetaminophen
Intracellular measurement of ATP-content (CellTiter-Glo®, Promega)
of 3D InSight™ Human Liver Microtissues after 72h exposure to ac50
etaminophen with daily medium renewal. Donor I shows an IC of
1093µM, donor II 2300µM. Error bars are standard deviation.
4.54
1
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
1
Fold-change vs. Control Sample
CD68
(A) H&E-staining and (B) CD68-staining of formalin-fixed, paraffin embedded 3D InSight™
Human Liver Microtissues over 28 days in
culture. H&E-staining indicates tight cell-cell
contacts and intact liver-like morphology.
Co-cultured CD68-positive Kupffer-cells integrate into Human Liver Microtissues and are
present throughout the culture period.
Concentration [uM]
# regulated proteins
Control
Day 28
Significantly regulated proteins
The numbers of significantly regulated proteins
(p=0.05, two-fold change) increase with acetaminophen concentration.
UDP-glucuronosyltransferase 1-1 (UniProt
P22309)
Enriched GO-terms in regulated proteins
DAVID functional annotation reveals relevant cellular processes that are activated or deactivated upon acetaminophen exposure. Numbers in
brackets are significance ranks.
9.77µM
Day 21
phen concentration range
2361 proteins were relatively quantified compared to the baseline
over the acetaminophen concentration range. Most proteins in 3D
Human Liver Microtissues don’t show induced changes in protein
expression and serve as baseline controls.
36.09µM
Day 14
H&E
Day 7
Targeted analysis of acetaminophen-relevant proteins
Acetaminophen at subtoxic concentrations is mainly metabolized via conjugation with sulfate or
glucuronide by sulfotransferases or glucuronosyltransferases. At high concentrations, these pathways become saturated and the highly reactive metabolite NAPQI is formed by cytochromes P450.
Generation of thousands of protein profiles over acetamino-
Concentration
Experimental summary
3D Human Liver Microtissues from two independent primary human hepatocyte lots (lot
IZT (donor I); S1195T (donor II) were co-cultured
with Kupffer cells (lot ZAR) and were exposed to
8 subtoxic concentrations of acetaminophen (4
µM - 3,310 µM) for 72 hours. Proteins from microtissues were extracted and subjected to mass
spectrometric analysis. Global proteome profiles
were generated using a library of over 2400 proteins. Treatment-dependent changes in protein
expression were determined by performing pairwise t-test comparisons of each acetaminophen
concentration with an untreated control.
Introduction
Early prediction of drug
safety remains a challenge in
preclinical phases of drug development. Global proteomic analysis
allows for a better understanding of the
mode of action and mechanism of toxicity induced by drug treatment. We applied
a novel mass-spectrometric approach - Hyper
Reaction Monitoring (HRM) – to quantify changes in protein expression in 3D InSight™ Human
Liver Microtissues exposed to acetaminophen.
Gene Name
CYP1A2
SRXN1
CYP2C8
CYP4F3
Downregulated
FoldChange
(Donor I/Donor II)
Function
Protein
4.5/10
NADPH-dependent electron
transport, aflatoxin B1 and acetaminophen metabolism
Fibrinogen alpha
chain
4.8/5.1
2.6/2.3
3.0/2.3
Gene Name
FGA
FN1
FoldChange
(Donor I/Donor II)
Function
-2.1/-2.8
Fibrin deposition / cofactor in
platelet aggregation (in blood)
-2.0/-2.2
Cell adhesion, cell motility,
wound healing
Oxidative stress resistance
Fibronectin
NADPH-dependent electron
transport, taxol metabolism
Hydroxymethylglutaryl-CoA synthase, cytoplasmic
HMGCS1
-4.5/-4.8
Intermediate biosynthesis, involved in liver development
Monooxygenase
Peroxisomal acyl-coenzyme A oxidase 1
ACOX1
-2.8/-2.8
Fatty acid metabolism
Conclusions
• Minimal sample quantity is required for large-scale proteomic study (12’000 cells/sample)
• Protein profiles across 48 samples from two donors were generated containing a total of more than 113,000 individual protein quantifications
• Identification of relevant proteins in acetaminophen metabolism (unbiased discovery approach)
• Targeted analysis of known proteins of interest allows for specific pathway analysis and reveals donor-specific expression patterns
• Global protein profiling using the novel mass spectrometric HRM approach enables quantifica-
tion of thousands of proteins over many states (treatments, time points, concentrations, etc.)

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