1 cell death induced by the phenolic antioxidant tert-butylhydroquinone and its metabolite...

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Cell death induced by the Cell death induced by the phenolic antioxidant phenolic antioxidant

terttert-butylhydroquinone -butylhydroquinone and its metabolite and its metabolite terttert-butylquinone in -butylquinone in

human monocytic human monocytic leukemia leukemia U937 cellsU937 cells

Okubo T, Yokoyama Y, Kano Okubo T, Yokoyama Y, Kano K, Kano I. K, Kano I.

Food and Chemical Food and Chemical Toxicology 2003; 41: 679-88Toxicology 2003; 41: 679-88

AdvisorAdvisor :Wantana :Wantana PhookongchanaPhookongchana

AdvisorAdvisor : Dr. Roongsiri : Dr. Roongsiri ChotpativategulChotpativategul

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IntroductionIntroduction

• Tert-butylhydroquinone (TBHQ) is a phenolic antioxidant used as food additive in oils, fat and meat product to prevent rancidity

• TBHQ’s metabolite is tert-butylquinone (TBQ)

• TBHQ and TBQ were cause DNA damage in forestomach epithelium of male F344 rats.

• Concentration of TBQ required to cause DNA damage was lower than TBHQ

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IntroductionIntroduction•Tert-butylsemiquinone anion radical is formed from TBHQ and TBQ and semiquinone dependent superoxide formation may contribute to the toxic actions

• Previous data demonstrated that TBHQ caused DNA cleavage in vitro

• TBHQ and TBQ have been reported to be cytotoxic in human lymphocyte , mechanism of cell death remain unclear

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Cell deathCell death

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ApoptosisApoptosis

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ObjectiveObjective

To investigate the pathways of cell death induced by TBHQ and TBQ by examining

caspase activities and various characteristic of cellular structure and function in human

monocytic leukemia U937cell, which is frequently used for cytotoxicity study

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Material & MethodMaterial & Method

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Cell culture Neutral red uptake assay Morphology of cells Flow cytometry Assay of caspase protease activities Western blotting of poly (ADP-ribose)

polymerase (PARP) protein Release of cytochrome c from mitochondria

to cytosol Determination of reduced glutathione Determination of cellular ATP level

Material and Material and MethodMethod

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Material and Material and MethodMethod

Cell cultureCell culture

U937

RPMI 1640 + 10% (v/v) fetal calf serum + antibiotics (gentamicin sulfate and kanamycin)

37 OC, 5% CO2

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Neutral red uptake Neutral red uptake assayassay

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Neutral red uptake assayNeutral red uptake assay

• Neutral red uptake is vital dye that accumulate in the lysosome of living cell.

• Death cell lose their ability to accumulate and retain neutral red

•Providing basis for simple colorimetric viability assay used widely in cellular toxicology.

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Neutral red uptake assayNeutral red uptake assay

1x105 cells/well

medium containing various concentration of TBHQ or TBQ

Absorbance 540 nm

RMPI1640 medium containing neutral red

1% acetic acid in 50% ethanol

24 h

3 h

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Neutral red uptake Neutral red uptake assayassay

ResultResultTBQ

TBHQ

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Morphology of cellMorphology of cell

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Fluorescence microscopy

Morphological change in apoptosis

Electron microscopy

Complex morphological change in apoptosis

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Morphology of cellMorphology of cell Fluorescence microscopyFluorescence microscopy

U937 cells

1.5 mM TBHQ 0.04 mM TBQ : 3 and 6 h

E/D (control) DMSO (control) : 3 hr

DAPI

Fluorescence microscope

DAPI; 4,6-diamidino-2-phenylindole dihydrochloride E/D ; ethanol/dodecane (98:2), DMSO ; dimetyl sulfoxide

U937 cells

DAPI

Fluorescence microscope

1.5 mM TBHQE/D (control)

0.04 mM TBQDMSO (control)

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Electron microscopyElectron microscopy

: 3 hr

: 3 hr


U937 cells

0.04 mM TBQ

DMSO(control) 1.5 mM TBHQ E/D (control)

uranyl acetate

TEM microscope

2 hr

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Morphology of cellsMorphology of cellsResultResult

Fluorescence microscopyFluorescence microscopy

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Morphology of cellsMorphology of cellsResultResult

Electron microscopyElectron microscopy

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Flow cytometryFlow cytometry

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To investigate disruption of mitochondrial transmembrane

potential

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1 h

RT, 20 min

•JC-1: 5,5'6,6'-tetrachloro-1,1',3,3'-tetra-ethylbenzimidazolcarbocyanine iodide

1.5 mM TBHQ

U937 cells

0.04 mM TBQ

FACScalibur flow cytometer

JC-1

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ResultResult

88%

85%83%

7% 65% 22%

Fig2.FACS analysis of mitochondrial membrane potential of cells stained with JC-1 after treat cells with 1.5 mM TBHQ or 0.04 mM TBQ for 1 h. Numbers refer to the percentage of cells encountered in each quadrant.

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Assay of caspase protease Assay of caspase protease activitiesactivities

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Assay of caspase Assay of caspase protease activitiesprotease activities

•Caspases are a family of proteins that are one of the main effectors of apoptosis.

•The caspases are a group of cysteine proteases that exist within the cell as inactive pro-forms or zymogens.

•These zymogens can be cleaved to form active enzymes following the induction of apoptosis.

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Enzyme activity

N-acetylcysteinemedium

-TBHQ

-TBQ

Lysis buffer

Cell lysate

2x106 cells/ml

GSH

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ResultResult

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Table1 Caspase activities induced by treatment of U937 cells with TBHQ and TBQ for 3 h

ResultResult

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Western blotting of Western blotting of poly (ADP-ribose) polymerase poly (ADP-ribose) polymerase

(PARP) protein(PARP) protein

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• Enzyme poly (ADP-ribose) polymerase, or PARP, was the first protein identified as a substrate for caspases.

•PARP is involved in repair of DNA damage and functions by catalyzing the synthesis of poly (ADP-ribose) and by binding to DNA strand breaks and modifying nuclear proteins.

•The ability of PARP to repair DNA damage is prevented following cleavage of PARP by caspase-3

Western blotting of poly Western blotting of poly (ADP-ribose) polymerase (ADP-ribose) polymerase

(PARP) protein(PARP) protein

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(PARP) protein(PARP) proteinU 937 cells

SDS–PAGE

Transfer to PVDF membrane

Anti-PARP antibody

Peroxidase-labeled goat antibody

Chemiluminescent reagents

Polaroid film

1.5 mMTBHQ 0.04 mM TBQ : 1.5, 3, 4.5, 6 h

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(PARP) protein(PARP) proteinResultResult

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Release of cytochrome cRelease of cytochrome c from mitochondria to cytosolfrom mitochondria to cytosol

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Cytochrome c is a protein that is important to the process of creating cellular energy, the main function of mitochondria. When mitochondria are damaged, cytochrome c is released into the main body of the cell, and if the cell itself is damaged, into surrounding tissue. The release of cytochrome c is part of the cascade of cellular events that lead to apoptosis

Release of cytochrome c Release of cytochrome c from mitochondria to from mitochondria to

cytosolcytosol

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U937 cells

E/D / 1.5 mM TBHQ / DMSO / 0.05 mMTBQ

Cytosolic extract

SDS-PAGE

Western blotting

Anti-cytochrome C antibody


cytosolcytosol

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cytosolcytosolResultResult

E/D TBHQ DMSO TBQ

Plate6. Release of cytochrome c detected by western blotting. Cells were treated with 105 mM TBHQ or 0.04 TBQ for 3 h.

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Determination of Determination of reduced glutathionereduced glutathione

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•Cellular glutathione which is an antioxidant contribute to defense against cell death.

•DEVDase activity was inhibited by glutathione

Determination of reduced Determination of reduced glutathioneglutathione

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U937 cells

TBHQ / TBQ

PBS

Cold perchlolic acid

O-phthalaldehyde

Cytofluor II

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resultresult

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Determination of Determination of cellular ATPcellular ATP

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Determination of cellular Determination of cellular ATPATP

U937 Cells

Glucose-free medium Glucose- containing medium

TBHQ / TBQ

Lysis buffer

Supernatant

Luminometer

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Determination of cellular Determination of cellular ATPATP

ResultResult

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DiscussionDiscussion

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DiscussionDiscussion

• Morphologically, nuclear condensation and fragmentation were observed time-dependently in TBHQ and TBQ treated cell.

• Breakdown of mitochondria transmembrane potential was suggested by decrease of JC-1 aggregation.

• Cytochrome c was released from mitochondria to cytosol by TBHQ or TBQ and DEVDase activity representing caspase-3,-7 was highly induced.

• Induction of DEVDase activity by TBHQ and TBQ was confirm by cleavage of PARP. •Induction of caspase activity by TBHQ and TBQ were prevented by addition of GSH and N-acetylcysteine

• ATP levels was decreased by the treatment of cells with TBHQ or TBQ.

1 cell death induced by the phenolic antioxidant tert-butylhydroquinone and its metabolite...

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