2. • There are many medications previously
marketed which cause hepatotoxicity;
constituting a public health problem
• Not able to predict if medications will have
toxic effects during development
- many medications get recalled after FDA
approval that are widely used
• By characterizing cells in vitro, we may be
able to find ways to predict clinical
hepatotoxicity
3. • During long term cell culture, hepatocyte cell
lines begin to degenerate, loss of morphology
and cell integrity
• Hepatocytes encounter loss of liver specific
functions (Ex: down regulation of phase I and
phase II enzyme activity)
• To fully characterize cells in vitro we need to
increase the viability of hepatocytes in culture
while maintaining cell morphology, integrity,
and function
4. • Acetaminophen is easily available and widely
used
- known to cause toxicity by conversion to
NAPQI by CYP450 enzymes
• Pierce et al. examined TAMH cells
- found that cells express enzymes homologous
to human CYP450 enzymes
- TAMH cells showed a dose related formation
of APAP-GSH metabolite via NAPQI production
5. • Alpha Mouse Liver (AML12) cells
• Produce transforming growth factor alpha
• Maintain epithelial morphology and distinctive
hepatocyte features for long periods in culture (Wu et
al, 1994)
• Express mRNA for hepatocyte markers (Wu et al,
1994)
• Are uncharacterized in regards to acetaminophen
response and use with plate coatings
• Little known about CYP450 characterization
6. • Cells in culture often lose liver specific functions
over time
• Cell culture methods are needed to prolong the
viability of cells in culture
- Collagen 1
- Matrigel
7. • AML12 Cells cultured on thin
coats of collagen or matrigel will
have more hepatocyte
characterization and sensitivity
to acetaminophen than cells
grown on non-coated tissue
culture plates
8. • Use three different tissue culture plate coatings
• Characterize morphology of the cells
- photographs
• Examine cell growth rates
-MTT
• Examine AML12 cell toxic response to APAP
-MTT
• Examine physiology (mRNA and protein expression)
-PCR
-Western Blot
9. • Cell Culture
• Preparation of Collagen Coated Plates
• Preparation of Matrigel Coated Plates
• RNA Harvesting/ RNA Isolation
• RT-PCR
• Protein Harvesting/ Western Blotting
• AML12 cell treatment with Acetaminophen
• MTT Assay
10. • Cell culture
- AML12 Complete Growth Media (Wu et al, 1994)
- All cultures maintained in a 37° C incubator with
5% CO2 (Coe, 2006, Wu et al, 1994)
- Passaged by trypsinization (Coe, 2006)
- Replating at ratios of 1:3 to 1:5 (Coe, 2006, Wu
et al, 1994)
- Initial seeding at 0.3 x 106 cells per 35mm plate
11. • Collagen Thin Coated Plates
- concentration of 50 mg/ml used in 0.02N Acetic Acid
- 1.6 ml of diluted collagen needed per 10cm2 of plate
surface area
- 100mm plates have a 60cm2 surface area so 10ml of
diluted collagen used to cover each plate
- one hour incubation at room temp. before rinsing
with HBSS
- once coated, cells passaged onto plates according to
standard protocols
12. • Matrigel Thin Coated Plates
- concentration 0f 0.3 mg/ml used in AML12
Complete Growth Media
- 2ml of diluted matrigel to coat plate surfaces
-one hour of drying time before aspiration of
excess liquid
- once coated, cells passaged onto plates
according to standard protocols
13. 1.) Growth Studies
- AML12 cells grown using the 3 different plate
treatments
-growth monitored for 5 days
- MTT assay completed once a day for 5 days,
every 24 hours
2.) Acetaminophen Toxicity
- AML12 cells grown using the 3 different plate
treatments
- treated with acetaminophen at concentrations
from 1 to 10 mM
- cells exposed for 36 hours before MTT
14. Figure 1: AML12 Cells Growth Monitoring
• AML12 cells on days 1 (A), 3 (B), and 5 (C) of growth
after sub-culturing on non-coated (1), matrigel coated
(2), and collagen coated (3) plates
15. Figure 2: AML12 Cell Growth
• Blue points are data from non-coated plates, red points are
data from collagen coated plates, and green points are data
from matrigel coated plates
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 1 2 3 4 5 6
AbsoluteAbsorbanceat595nm
Time (Days)
16. • Summary of Analysis
- Use of different plate coatings did not cause
any differences to occur as predicted
- Neither the growth rate of the cells, overall
cell number, nor cell morphology was
observed to show differences between the
three plate coatings used
-No significant differences between coatings
17. • Results of the analysis were not as expected
• Possible Limitations:
1.) Initial cell seeding was at too high of a
density
2.) Concentrations of coatings used
18. Figure 3: Observable Toxicity of AML12 Cells
• 0 mM (A), 5 mM (B), and 10 mM (C) after sub-culturing
on non-coated (1), matrigel coated (2), and collagen coated
(3) plates
20. Figure 5: AML12 Drug Toxicity
0
20
40
60
80
100
120
140
CellViabilityComparedtoControl
Drug Treatment (1 mM)
21. • Summary of analysis
- No discernible physical changes could be observed in
the AML12 cells upon exposure to acetaminophen
- Acetaminophen did not cause toxicity to the AML12
cells based upon results of the MTT analysis
- Toxic effects of acetaminophen were not greater in
AML12 cells grown on collagen and matrigel coated
plates
- Significant differences observed between plate
coatings
22. • Results were not as expected
• Possible limitations
1.) Cell passage number
2.) Miscalculation of doses
3.) Concentrations of acetaminophen used were not close
enough to LC50 for AML12 cells
4.) Acetaminophen never underwent the toxic metabolic
pathway
5.) Too short of an exposure time
23. • Harvesting of RNA/RNA Isolation
-Trizol harvesting of lysates and Trizol Protocol
(Invitrogen)
- Jasco spectrophotometer and Spectra Manager
program
-A260/280 ratio
Absorbance at 260nm x 40 x100/ 1000 = mg/ml RNA
- RNA used to make cDNA using qScript cDNA Synthesis
Kit
25. Figure 6: PCR Optimization
• PCR optimization for primers for albumin (lane2), actin (lane 3),
CYP1A1 (lane 4), CYP2E1 (lane 5), and UGT2B35 (lane 6). The
annealing temperature was 58 °C for 40 cycles.
26. Table 2: RT-PCR Parameters
• PCR products analyzed by 1% agarose gel
electrophoresis
PCR Parameters
Primer Pair Denaturation Cycles Annealing Temp Extension Final Extension
Actin 95° C for 5 min 30 58°C 72° C for 60 s 72° C for 5 min
Albumin 95° C for 5 min 30 58°C 72° C for 60 s 72° C for 5 min
UGT2B35 95° C for 5 min 30 58°C 72° C for 60 s 72° C for 5 min
CYP1A1 95° C for 5 min 40 57°C 72° C for 60 s 72° C for 5 min
CYP2E1 95° C for 5 min 40 59°C 72° C for 60 s 72° C for 5 min
27. Figure 7: PCR Optimization Rf Calculation Gels
A.) B.) C.)
- Bands produced were used for Rf calculation band
confirmation measurements
28. Table 3: Rf Calculation Data
-PCR product sizes were able to be confirmed
Rf Calculation
Primer Pair Expected Product Size Length of Band (mm) Plot Equation R Squared Calculated bp
Actin 294 bp 0.6556
y= -2.2295x + 3.9621 0.9876
317
Albumin 234 bp 0.7000 252
UGT2B35 186 bp 0.8444 y= -1.8009 + 3.8021 0.9983 191
CYP1A1 306 bp 2.4420
y = -2.2329x + 3.7929 0.9984
277
CYP2E1 312 bp 2.5313 340
29. Figure 8: RT-PCR Results
A.) B.) C.)
• Non-coated: actin (lane 2), albumin (lane 3), UGT2B35 (lane 4), CYP2E1 (lane
5), CYP1A1 (lane 6); collagen coated: actin (lane 8), albumin (lane 9),
UGT2B35 (lane 10), CYP2E1 (lane 11), CYP1A1 (lane 12); matrigel coated:
actin (lane 14), albumin (lane 15), UGT2B35 (lane 16), CYP2E1 (lane 17), and
CYP1A1 (lane 18)
30. Figure 9: Compared mRNA Expression
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Non-coated Collagen Matrigel
RelativemRNAExpression
Plate Treatments
• AML12 mRNA expression from each primer pair: actin (blue), albumin (red),
UGT2B35 (green), CYP2E1 (purple), and CYP1A1 (turquoise) based on the RT-PCR
results from cells grown on non-coated plates, matrigel coated plates, and
collagen coated plates
31. • Summary of Analysis
- AML12 cells grown on collagen plates showed
mRNA expression for only actin and albumin
- Albumin expression of cells on collagen plates
did surpass that of cells on non-coated plates
- AML12 cells grown on matrigel coated plates
showed mRNA expression for all proteins and
enzymes, but expression did not surpass that
exhibited by cells on non-coated plates
- No statistical differences between plate coatings
32. • Results were not as expected
• Possible limitations
1.) Uneven number of cells on plates harvested
for mRNA
2.) Concentration of matrigel too low to effect
mRNA expression
3.) Concentration of collagen too high
33. • Samples harvested from AML12 cells plated using
the 3 different culture methods using Llaemmli
Buffer
• SDS-PAGE used to separate proteins by their
molecular weights
• 10% polyacrylamide gel
• Membrane probed with antibodies to actin,
albumin, CYP1A1, and CYP2E1 (Thermo Scientific and
Abcam)
• SuperSignal West Pico (Thermo Scientific) substrate
for detection
34. Figure 10: Western Blot
• Protein samples harvested from non-coated plates (lane 1 -4); collagen coated plates
(lane 5-8) ; and matrigel coated plates (lane 9-12) . Four different sample sizes were
used:10 µl (lanes 1,5, and 9); 20 µl (lanes 2, 6, and 10), 30 µl (lanes 3, 7, and 11); and 40 µl
(lanes 4, 8, and 12). The blot was probed with an antibody for CYP1A1.
35. • When probed for CYP1A1 no specific bands
were produced on the blot
• Analysis was repeated for albumin and
CYP2E1
• None of the analytes tested produced specific
bands on the western blots attempted
36. • Results were not as expected
• Possible limitations
1.) Antibodies were incorrect and not able to
detect for the specific proteins monitored
during the analysis
2.) Protein samples were saturated with
proteins from collagen and matrigel coatings
that masked the specific proteins needed
37. • Hypothesis was rejected
• Use of collagen and matrigel extracellular
matrices did not have any enhancing effects on
cell viability in culture, cell morphology,
sensitivity to acetaminophen, or mRNA
expression
• For the purposes of this study AML12 cell lines did
not prove to be a good hepatocyte model
• However, the study did allow us to examine a
hepatocyte cell line that is not well known
38. • Use varying concentrations of collagen and
matrigel to determine optimal concentration
for these cells
• Explore lower passages of AML12 cells to see
if passage number effects growth and drug
sensitivity
• Use a broader range of acetaminophen doses
on the AML12 cells to pinpoint an LD50 or
LC50
39. • Take MTT absorbance readings every 24
hours up to final time point to determine
optimal exposure to cause toxicity in AML12
cells
• HPLC analysis for GSH acetaminophen
conjugate
• Obtain new antibodies for western blot
• Isolating cellular microsomal fractions for
western blot analysis
40. • Dr. Adams, Dr. Bloom, and Dr. Abraham for being my
mentors and helping me through the duration of the
project
• Dr. Al-Achi for help with utilizing JMP software
• Dr. Breivogel for helping me with the mice and
advisement for the duration of my years here at CU
• Chad Moody for help with lab procedures, techniques,
and general support
• An endless list of faculty, friends, and family for
numerous types of support throughout the
completion of my coursework
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