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

Thesis DefenseThesis Defense

April 23. 2007April 23. 2007

Yeong-Nam JeongYeong-Nam Jeong

Marshall UniversityMarshall University

History of ArsenicHistory of Arsenic

Used over 2,400 years for medical Used over 2,400 years for medical purposes purposes

In the 18th century: used therapeutically In the 18th century: used therapeutically Fowler’s solution: 1% arsenic trioxide in Fowler’s solution: 1% arsenic trioxide in

potassium bicarbonate, major therapy for potassium bicarbonate, major therapy for leukemia treatment leukemia treatment

In the 19th century: In the 19th century: materia medicamateria medica In the 1970s: Chinese researcher studied In the 1970s: Chinese researcher studied

as a treatment for acute promyelocytic as a treatment for acute promyelocytic leukemia (APL) leukemia (APL)

ArsenicArsenic

Two main categories of compoundsTwo main categories of compounds OrganicOrganic InorganicInorganic

Organic arsenic compounds are Organic arsenic compounds are combined with Hydrogen and Carboncombined with Hydrogen and Carbon

Inorganic arsenic compounds contain Inorganic arsenic compounds contain Oxygen, Chlorine, and Sulfur. Oxygen, Chlorine, and Sulfur.

Inorganic arsenic is more harmful than Inorganic arsenic is more harmful than organic arsenic.organic arsenic.

http://www.uky.edu/WaterResources/SYMP01-ITAC.HTMLhttp://www.uky.edu/WaterResources/SYMP01-ITAC.HTML

Arsenic is ToxicArsenic is Toxic

Arsenic has been linked to several forms of Arsenic has been linked to several forms of cancercancer

Arsenic is associated with heart, lung, Arsenic is associated with heart, lung, immunological, nerve and hormone problems. immunological, nerve and hormone problems.

Inorganic arsenic has both acute and chronic Inorganic arsenic has both acute and chronic toxic effects.toxic effects.

How arsenic causes cancer is not well How arsenic causes cancer is not well understood. understood.

Apoptosis may be involved.Apoptosis may be involved.

http://www.epa.gov/watersecurity/guide/chemicalsensorforarsenic.htmlhttp://www.epa.gov/watersecurity/guide/chemicalsensorforarsenic.html

PLHC-1 Cell linesPLHC-1 Cell lines

Poeciliopsis lucidaPoeciliopsis lucida hepatocellular hepatocellular carcinoma carcinoma

Derived from a liver tumor from topminnow Derived from a liver tumor from topminnow Used to screen heavy metals and other Used to screen heavy metals and other

environmental toxins using a combined environmental toxins using a combined stress protein and cytotoxicity assay.stress protein and cytotoxicity assay.

Comet Assay – DNA Damage

DNA fragmentation assayDNA fragmentation assay

Monitor apoptosis using PLHC-1 cells induced Monitor apoptosis using PLHC-1 cells induced by Asby As22OO33 and Cadmium Chloride and Cadmium Chloride

ApoptosisApoptosis Nuclear chromatin condensationNuclear chromatin condensation Shrinkage of cellsShrinkage of cells Disintegration of nuclear membraneDisintegration of nuclear membrane Break down of plasma membrane and formation of Break down of plasma membrane and formation of

membrane-bound broken down cells (apoptotic membrane-bound broken down cells (apoptotic bodies)bodies)

Degraded nuclear DNA into ‘DNA ladder’ (Wyllie, Degraded nuclear DNA into ‘DNA ladder’ (Wyllie, 1981).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 MethodsMaterials and Methods

Cells were grown in T-75 cmCells were grown in T-75 cm22 flasks at 30 flasks at 30ooC COC CO2 2

Incubator in Minimum essential medium (Eagle) Incubator in Minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium non-essential amino acids, and 1.0 mM sodium pyruvate, 95%; fetal bovine serum, 5% and 1% pyruvate, 95%; fetal bovine serum, 5% and 1% penicillin streptomycin.penicillin streptomycin.

Cadmium Chloride 0.1M solution 1, 2 and 10 Cadmium Chloride 0.1M solution 1, 2 and 10 M M was prepared for cometwas prepared for comet

Results: Cell Growth Assay IResults: Cell Growth Assay I

Results: Cell Growth Assay IIResults: Cell Growth Assay II

Results: Cell Growth Assay IIIResults: Cell Growth Assay III

MaterialsMaterials

AsAs22OO55 – Arsenic (V) oxide, 99.9% (metal – Arsenic (V) oxide, 99.9% (metal

basis), Packed Under Argon. Stock basis), Packed Under Argon. Stock #14668, Lot #L06M05. Alfa Aesar#14668, Lot #L06M05. Alfa Aesar

AsAs22OO33 – Arsenic (III) oxide, 99.9% (metal – Arsenic (III) oxide, 99.9% (metal

basis), Packed Under Argon. Stock basis), Packed Under Argon. Stock #40370, Lot #M19I12. Alfa Aesar#40370, Lot #M19I12. Alfa Aesar

AsAs22OO33 AsAs

22OO55 Cadmium Cadmium ChlorideChloride

1 1 MM 1 1 MM 1 1 MM

5 5 MM 5 5 MM 2 2 MM

10 10 MM 10 10 MM 10 10 MM

Experimental Concentrations

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 ScoringCategorical Scoring

Categorical Data is SignificantCategorical Data is Significant

Comet Assay IV SoftwareComet Assay IV Software

Two close cells were not good to Two close cells were not good to measure.measure.

An example of a round, An example of a round, undamaged cellsundamaged cells

An example with medium damaged An example with medium damaged cellscells

An example with high damaged An example with high damaged cellscells

AS2O3CON

10.005.001.00.00

Mea

n H

L21.5

21.0

20.5

20.0

19.5

19.0

18.5

18.0

17.5

TIME

1.00

2.00

Head Length Mean Graph with As2O3 concentration

AS2O3CON

10.005.001.00.00

Mea

n T

L40

30

20

10

TIME

1.00

2.00

Tail Length Mean Graph with As2O3 concentration

AS2O3CON

10.005.001.00.00

Mea

n H

I90

80

70

60

TIME

1.00

2.00

Head Intensity Mean Graph with As2O3 concentration

AS2O3CON

10.005.001.00.00

Mea

n T

I30

20

10

TIME

1.00

2.00

Tail Intensity Mean Graph with As2O3 concentration

AS2O3CON

10.005.001.00.00

Mea

n T

M8

7

6

5

4

3

2

1

TIME

1.00

2.00

Tail Moment Mean Graph with As2O3 concentration

AS2O5CON

10.005.001.00.00

Mea

n T

M8

7

6

5

4

3

2

TIME

1.00

2.00

Tail Moment Mean Graph with As2O5 concentration

Why Does DNA Laddering Occur?Why Does DNA Laddering Occur?

http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/nuclear.html

DNA Laddering AssayDNA Laddering Assay

As2O3 Cadmium Chloride

Normal appearance of cultured Normal appearance of cultured PHLC-1 cellsPHLC-1 cells

The FragELThe FragELTMTM DNA fragmentation Detection DNA fragmentation Detection Kit, Fluorescent TdT Enzyme Kit, Fluorescent TdT Enzyme from from

CalBiochemCalBiochem

Enzymatic addition of labeled Enzymatic addition of labeled nucleotides was carried out by using nucleotides was carried out by using Terminal Deoxynucleotidyl Terminal Deoxynucleotidyl Transferase (TdT) and fluorescein Transferase (TdT) and fluorescein labeled deoxynucleotides. labeled deoxynucleotides.

The Bio-Rad MRC1024 Confocal The Bio-Rad MRC1024 Confocal Scanning Microscope Scanning Microscope

No arsenic treatment control cells. No arsenic treatment control cells.

55M AsM As22OO33 1hr 1hr

55M AsM As22OO3 3 3hr 3hr

Future DirectionsFuture Directions Comet Assay: Controls have background Comet Assay: Controls have background

problems and it could be trypsin or cell growth problems and it could be trypsin or cell growth pattern. pattern.

DNA ladder assay: PLHC-1 may have different DNA ladder assay: PLHC-1 may have different pathwaypathway

MTT assay: I am repeating this assay for my MTT assay: I am repeating this assay for my class, if it works I will add to the thesis.class, if it works I will add to the thesis.

Western blot: I am repeating this assay for my Western blot: I am repeating this assay for my class, if it works I will add to the thesis. class, if it works I will add to the thesis.

Other experiments: DAPI, HSP-70, Caspase, Other experiments: DAPI, HSP-70, Caspase, TUNEL assay etc..TUNEL assay etc..

ConclusionsConclusions

PLHC-1 cells can be useful for comet PLHC-1 cells can be useful for comet assayassay

AsAs22OO33 causes DNA damages in PLHC-1 causes DNA damages in PLHC-1 cells a dose dependent fashion. 1 hour cells a dose dependent fashion. 1 hour had better data in comet assay than 2 had better data in comet assay than 2 hours because cells were more damaged hours because cells were more damaged after 2 hours.after 2 hours.

AsAs22OO5 5 also cause DNA damage but it was also cause DNA damage but it was less consistent than Asless consistent than As22OO33

ConclusionsConclusions

AsAs22OO3 3 did not cause DNA laddering in PLHC-1 did not cause DNA laddering in PLHC-1

cells.cells. Camptothecin and Cadmium also did not cause Camptothecin and Cadmium also did not cause

DNA laddering in PLHC-1 cells.DNA laddering in PLHC-1 cells. Wang Wang et alet al. shows that some fish cells do not . shows that some fish cells do not

have DNA laddering during apoptosis.have DNA laddering during apoptosis. The FragELThe FragELTMTM DNA fragmentation Detection Kit DNA fragmentation Detection Kit

Assay was positive for apoptosis DNA damages Assay was positive for apoptosis DNA damages with PLHC-1 cell in Aswith PLHC-1 cell in As22OO3 3 treatment.treatment.

AcknowledgementsAcknowledgements Dr. Collier for using Olympus BX51 microscope and Dr. Collier for using Olympus BX51 microscope and

photomicrographs were take at using Olympus photomicrographs were take at using Olympus Microsuite™ Basic software. Microsuite™ Basic software.

Mr. David Neff for using Confocal Microscope and Mr. David Neff for using Confocal Microscope and The The FragELFragELTMTM DNA fragmentation Detection Kit DNA fragmentation Detection Kit Assay . Assay .

Dr. Cohenford for fluorimeter.Dr. Cohenford for fluorimeter. Mr. Pete Glass and Dr. Little for GIS class.Mr. Pete Glass and Dr. Little for GIS class. Dr. Harrison for using hemocytometer. Dr. Harrison for using hemocytometer. IST 343 student for help with MTT assay.IST 343 student for help with MTT assay. Ms. Wanda Dyke for help with all ordering, receiving, PAR Ms. Wanda Dyke for help with all ordering, receiving, PAR

and so on.and so on. Dr. Murray and Committee for reading, writing and Dr. Murray and Committee for reading, writing and

presentation help.presentation help. Thanks to My family for sending me to US, especially Thanks to My family for sending me to US, especially

Jooha Jeong. Jooha Jeong.

Thank you…

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