Cell death induced by the phenolic antioxidant
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Okubo T, Yokoyama Y, Kano K, Kano I. Food and Chemical Toxicology 2003; 41: 679-88 PowerPoint PPT Presentation


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Cell death induced by the phenolic antioxidant tert -butylhydroquinone and its metabolite tert -butylquinone in human monocytic leukemia U937 cells. Okubo T, Yokoyama Y, Kano K, Kano I. Food and Chemical Toxicology 2003; 41: 679-88 Advisor :Wantana Phookongchana

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Okubo T, Yokoyama Y, Kano K, Kano I. Food and Chemical Toxicology 2003; 41: 679-88

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Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Cell death induced by the phenolic antioxidant tert-butylhydroquinone and its metabolite tert-butylquinone in human monocytic leukemia U937 cells

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

Food and Chemical Toxicology 2003; 41: 679-88

Advisor:Wantana Phookongchana

Advisor: Dr. Roongsiri Chotpativategul


Introduction

Introduction

  • 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


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Introduction

  • 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


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Cell death


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Apoptosis


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Objective

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


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Material & Method


Material and method

Material and Method

  • 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 method1

Material and Method

Cell culture

U937

RPMI 1640 + 10% (v/v) fetal calf serum +

antibiotics (gentamicin sulfate and kanamycin)

37 OC, 5% CO2


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Neutral red uptake assay


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Neutral 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.


Neutral red uptake assay

Neutral red uptake assay

1x105 cells/well

medium containing various concentration of TBHQ or TBQ

24 h

RMPI1640 medium containing neutral red

3 h

1% acetic acid in 50% ethanol

Absorbance 540 nm


Neutral red uptake assay1

Neutral red uptake assay

Result

TBQ

TBHQ


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Morphology of cell


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Morphology of cell

Morphological change in apoptosis

Fluorescence microscopy

Complex morphological change in apoptosis

Electron microscopy


Morphology of cell

Morphology of cell

Fluorescence 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

1.5 mM TBHQ

E/D (control)

0.04 mM TBQ

DMSO (control)

DAPI

Fluorescence microscope


Morphology of cell1

Morphology of cell

Electron microscopy

: 3 hr

: 3 hr

U937 cells

0.04 mM TBQ

DMSO(control)

1.5 mM TBHQ

E/D (control)

uranyl acetate

2 hr

TEM microscope


Morphology of cells

Morphology of cells

ResultFluorescence microscopy


Morphology of cells1

Morphology of cells

ResultElectron microscopy


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Flow cytometry


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Flow cytometry

To investigate disruption of mitochondrial transmembrane potential


Flow cytometry

Flow cytometry

U937 cells

1 h

RT, 20 min

1.5 mM TBHQ

0.04 mM TBQ

JC-1

FACScalibur flow cytometer

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


Flow cytometry1

Flow cytometry

Result

7%

65%

22%

88%

83%

85%

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.


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Assay of caspase protease

activities


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Assay of caspase protease 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.


Assay of caspase protease activities

Assay of caspase protease activities

2x106 cells/ml

N-acetylcysteine

medium

GSH

-TBHQ

-TBQ

Lysis buffer

Cell lysate

Enzyme activity


Assay of caspase protease activities1

Assay of caspase protease activities

Result


Assay of caspase protease activities2

Assay of caspase protease activities

Result

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


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Western blotting of

poly (ADP-ribose) polymerase (PARP) protein


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

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

  • 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 adp ribose polymerase parp protein

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

U 937 cells

SDS–PAGE

Transfer to PVDF membrane

Anti-PARP antibody

Peroxidase-labeled goat antibody

Chemiluminescent reagents

Polaroid film

Method

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


Western blotting of poly adp ribose polymerase parp protein1

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

Result


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Release of cytochrome c

from mitochondria to cytosol


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Release of cytochrome c from mitochondria to cytosol

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 from mitochondria to cytosol

Release of cytochrome c from mitochondria to cytosol

Method

U937 cells

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

Cytosolic extract

SDS-PAGE

Western blotting

Anti-cytochrome C antibody


Release of cytochrome c from mitochondria to cytosol1

Release of cytochrome c from mitochondria to cytosol

Result

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.


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Determination of

reduced glutathione


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Determination of reduced glutathione

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

  • DEVDase activity was inhibited by glutathione


Determination of reduced glutathione

Determination of reduced glutathione

Method

U937 cells

TBHQ / TBQ

PBS

Cold perchlolic acid

O-phthalaldehyde

Cytofluor II


Determination of reduced glutathione1

Determination of reduced glutathione

result


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Determination of

cellular ATP


Determination of cellular atp

Determination of cellular ATP

U937 Cells

Glucose- containing medium

Glucose-free medium

TBHQ / TBQ

Lysis buffer

Supernatant

Luminometer


Determination of cellular atp1

Determination of cellular ATP

Result


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Discussion


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Discussion

  • 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.


Okubo t yokoyama y kano k kano i food and chemical toxicology 2003 41 679 88

Thank you for your attention


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