Download The Effects of Arsenic Toxicity in PLHC-1 Cell Line

The Effects of Arsenic
Toxicity in PLHC-1 Cell Line
Thesis Defense
April 23. 2007
Yeong-Nam Jeong
Marshall University
History of Arsenic
 Used
over 2,400 years for medical
purposes
 In the 18th century: used therapeutically
 Fowler’s solution: 1% arsenic trioxide in
potassium bicarbonate, major therapy for
leukemia treatment
 In the 19th century: materia medica
 In the 1970s: Chinese researcher studied
as a treatment for acute promyelocytic
leukemia (APL)
Arsenic
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Two main categories of compounds
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
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Organic
Inorganic
Organic arsenic compounds are
combined with Hydrogen and Carbon
Inorganic arsenic compounds contain
Oxygen, Chlorine, and Sulfur.
Inorganic arsenic is more harmful than
organic arsenic.
http://www.uky.edu/WaterResources/SYMP01-ITAC.HTML
Arsenic is Toxic
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Arsenic has been linked to several forms of
cancer
Arsenic is associated with heart, lung,
immunological, nerve and hormone problems.
Inorganic arsenic has both acute and chronic
toxic effects.
How arsenic causes cancer is not well
understood.
Apoptosis may be involved.
http://www.epa.gov/watersecurity/guide/chemicalsensorforarsenic.html
PLHC-1 Cell lines
 Poeciliopsis
lucida hepatocellular
carcinoma
 Derived from a liver tumor from topminnow
 Used to screen heavy metals and other
environmental toxins using a combined
stress protein and cytotoxicity assay.
Comet Assay – DNA Damage
DNA fragmentation assay

Monitor apoptosis using PLHC-1 cells induced
by As2O3 and Cadmium Chloride
 Apoptosis
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Nuclear chromatin condensation
Shrinkage of cells
Disintegration of nuclear membrane
Break down of plasma membrane and formation of
membrane-bound broken down cells (apoptotic
bodies)
Degraded nuclear DNA into ‘DNA ladder’ (Wyllie,
1981).
This picture shows that arsenite-treated TO-2 cell
line has no DNA ladder and arsenite-treated JF
cell line detect apoptosis (Wang et al. 2004).
Materials and Methods
Cells were grown in T-75 cm2 flasks at 30oC CO2
Incubator in Minimum essential medium (Eagle)
with 2 mM L-glutamine and Earle's BSS
adjusted to contain 1.5 g/L sodium bicarbonate,
0.1 mM non-essential amino acids, and 1.0 mM
sodium pyruvate, 95%; fetal bovine serum, 5%
and 1% penicillin streptomycin.
 Cadmium Chloride 0.1M solution 1, 2 and 10 mM
was prepared for comet

Results: Cell Growth Assay I
Results: Cell Growth Assay II
Results: Cell Growth Assay III
Materials
– Arsenic (V) oxide, 99.9% (metal
basis), Packed Under Argon. Stock
#14668, Lot #L06M05. Alfa Aesar
 As2O3 – Arsenic (III) oxide, 99.9% (metal
basis), Packed Under Argon. Stock
#40370, Lot #M19I12. Alfa Aesar
 As2O5
Experimental Concentrations
As2O3
As2O5
Cadmium
Chloride
1 mM
1 mM
1 mM
5 mM
5 mM
2 mM
10 mM
10 mM
10 mM
Arsenic Type
Day
Time of Exposure
hours
Treated Wells
As2O3
1
1
1
As2O3
1
1
2
As2O3
1
2
1
As2O3
1
2
2
As2O3
2
1
1
As2O3
2
1
2
As2O3
2
2
1
As2O3
2
2
2
As2O5
1
1
1
As2O5
1
1
2
As2O5
1
2
1
As2O5
1
2
2
As2O5
2
1
1
As2O5
2
1
2
As2O5
2
2
1
As2O5
2
2
2
Categorical Scoring
Categorical Data is Significant
Comet Assay IV Software
Two close cells were not good to
measure.
An example of a round,
undamaged cells
An example with medium damaged
cells
An example with high damaged
cells
Head Length Mean Graph with As2O3 concentration
21.5
21.0
20.5
20.0
19.5
19.0
TIME
Mean HL
18.5
18.0
1.00
17.5
2.00
.00
AS2O3CON
1.00
5.00
10.00
Tail Length Mean Graph with As2O3 concentration
40
30
20
Mean TL
TIME
1.00
10
2.00
.00
AS2O3CON
1.00
5.00
10.00
Head Intensity Mean Graph with As2O3 concentration
90
80
70
Mean HI
TIME
1.00
60
2.00
.00
AS2O3CON
1.00
5.00
10.00
Tail Intensity Mean Graph with As2O3 concentration
30
20
Mean TI
TIME
1.00
10
2.00
.00
AS2O3CON
1.00
5.00
10.00
Tail Moment Mean Graph with As2O3 concentration
8
7
6
5
4
Mean TM
3
TIME
2
1.00
1
2.00
.00
AS2O3CON
1.00
5.00
10.00
Tail Moment Mean Graph with As2O5 concentration
8
7
6
5
4
Mean TM
TIME
3
1.00
2
2.00
.00
AS2O5CON
1.00
5.00
10.00
Why Does DNA Laddering Occur?
http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/nuclear.html
DNA Laddering Assay
As2O3
Cadmium Chloride
Normal appearance of cultured
PHLC-1 cells
The FragELTM DNA fragmentation Detection
Kit, Fluorescent TdT Enzyme from
CalBiochem
 Enzymatic
addition of labeled
nucleotides was carried out by using
Terminal Deoxynucleotidyl
Transferase (TdT) and fluorescein
labeled deoxynucleotides.
 The Bio-Rad MRC1024 Confocal
Scanning Microscope
No arsenic treatment control cells.
5mM As2O3 1hr
5mM As2O3 3hr
Future Directions
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Comet Assay: Controls have background
problems and it could be trypsin or cell growth
pattern.
DNA ladder assay: PLHC-1 may have different
pathway
MTT assay: I am repeating this assay for my
class, if it works I will add to the thesis.
Western blot: I am repeating this assay for my
class, if it works I will add to the thesis.
Other experiments: DAPI, HSP-70, Caspase,
TUNEL assay etc..
Conclusions
 PLHC-1
cells can be useful for comet
assay
 As2O3 causes DNA damages in PLHC-1
cells a dose dependent fashion. 1 hour
had better data in comet assay than 2
hours because cells were more damaged
after 2 hours.
 As2O5 also cause DNA damage but it was
less consistent than As2O3
Conclusions

As2O3 did not cause DNA laddering in PLHC-1
cells.
 Camptothecin and Cadmium also did not cause
DNA laddering in PLHC-1 cells.
 Wang et al. shows that some fish cells do not
have DNA laddering during apoptosis.
 The FragELTM DNA fragmentation Detection Kit
Assay was positive for apoptosis DNA damages
with PLHC-1 cell in As2O3 treatment.
Acknowledgements
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Dr. Collier for using Olympus BX51 microscope and
photomicrographs were take at using Olympus
Microsuite™ Basic software.
Mr. David Neff for using Confocal Microscope and The
FragELTM DNA fragmentation Detection Kit Assay .
Dr. Cohenford for fluorimeter.
Mr. Pete Glass and Dr. Little for GIS class.
Dr. Harrison for using hemocytometer.
IST 343 student for help with MTT assay.
Ms. Wanda Dyke for help with all ordering, receiving,
PAR and so on.
Dr. Murray and Committee for reading, writing and
presentation help.
Thanks to My family for sending me to US, especially
Jooha Jeong.
Thank you…
References
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15.
16.
Samuel Waxman, Kenneth C. Anderson. History of the Development of Arsenic Derivatives in Cancer
Therapy. Oncologist. 2001; 6: 3-10
http://www.uky.edu/WaterResources/SYMP01-ITAC.HTML
http://www.wise-uranium.org/mdafin.html
http://www.epa.gov/watersecurity/guide/chemicalsensorforarsenic.html
Jin Y, Sun G, Li X, Li G, Lu C, Qu L. Study on the toxic effects induced by different arsenicals in primary
cultured rat astroglia. Toxicol Appl Pharmacol. 2004; 196:396-403.
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki
YF. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol
Mutagen. 2000; 35:206-21.
C. Risso-de Faverney, A. Devaux, M. Lafaurie, J.P Girard. B. Bailly, R. Rahmani. Cadmium induces apoptosis
and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species. Aquatic
Toxicology. 2001; 53: 65-76
Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA
damage in individual cells. Exp Cell Res. 1988 Mar; 175(1):184-91.
Ryan JA, Hightower LE. Evaluation of heavy-metal ion toxicity in fish cells using a combined stress protein and
cytotoxicity assay. Environ. Tox. Chem 1994; 13: 1231-1240.
Krone PH, Blechinger SR, Evans TG, Ryan JA, Noonan EJ, Hightower LE. Use of fish liver PLHC-1 cells and
zebrafish embryos in cytotoxicity assays. Methods. 2005; 35:176-87.
www.nativefish.org/Gallery
Xin-Mei Liu, Jian-Zhong Shao, Li-Xin Xian, Xian-Yong Chen. Cytotoxic Effect and Apoptosis Induction of
Atrazine in a Grass Carp (Ctenopharyngodon idellus) Cell Line. WILEY InterScience. 2006. 80-89.
Zigang D. The Molecular Mechanisms of Arsenic-Induced Cell Transformation and Apoptosis. Environmental
Health Perspectives. 2002; 110(5): 757-759.
Yu-Chieh Wang, Ren-Haw Chaung, Li-Chu Tung. Comparison of the cytotoxicity induced by different exposure
to sodium arsenite in two fish cell lines. Aquatic Toxicology. 2004; 69: 67-79
M Rau Embry, SM Billiard, RT Di Giulio. Lack of p53 induction in fish cells by model chemotherapeutics.
Oncogene. 2006; 25: 2004-2010.
R. Ian Freshney. Culture of Animal Cells. A Manual of Basic Technique. 4 th Edition. Wiley-Liss