The Effects of Arsenic Toxicity in PLHC1 Cell Line

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The Effects of Arsenic Toxicity in PLHC-1 Cell
Line

• Thesis Defense
• April 23. 2007
• Yeong-Nam Jeong
• Marshall University

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History of Arsenic

• Used over 2,400 years for medical purposes
• In the 18th century used therapeutically
• Fowlers 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)

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Arsenic

• Two main categories of compounds
• 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

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Arsenic is Toxic

• 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/chemicalsen
  sorforarsenic.html

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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.

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Comet Assay DNA Damage
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DNA fragmentation assay

• Monitor apoptosis using PLHC-1 cells induced by
  As2O3 and Cadmium Chloride
• Apoptosis
• 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).

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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).
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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

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Results Cell Growth Assay I
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Results Cell Growth Assay II
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Results Cell Growth Assay III
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Materials

• As2O5 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

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Experimental Concentrations
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Categorical Scoring
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Categorical Data is Significant
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Comet Assay IV Software
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Two close cells were not good to measure.
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An example of a round, undamaged cells
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An example with medium damaged cells
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An example with high damaged cells
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Head Length Mean Graph with As2O3 concentration
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Tail Length Mean Graph with As2O3 concentration
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Head Intensity Mean Graph with As2O3 concentration
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Tail Intensity Mean Graph with As2O3 concentration
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Tail Moment Mean Graph with As2O3 concentration
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Tail Moment Mean Graph with As2O5 concentration
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Why Does DNA Laddering Occur?
http//www.sgul.ac.uk/depts/immunology/dash/apopt
osis/nuclear.html
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DNA Laddering Assay
As2O3
Cadmium Chloride
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Normal appearance of cultured PHLC-1 cells
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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

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No arsenic treatment control cells.
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5mM As2O3 1hr
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5mM As2O3 3hr
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Future Directions

• 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..

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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

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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.

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Acknowledgements

• 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.

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Thank you
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