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

1. 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

2. 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

3. 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

4. Cell death

5. Apoptosis

6. 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

7. Material & Method

8. 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

9. Material and Method Cell culture U937 RPMI 1640 + 10% (v/v) fetal calf serum + antibiotics (gentamicin sulfate and kanamycin) 37 OC, 5% CO2

10. Neutral red uptake assay

11. 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.

12. 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

13. Neutral red uptake assay Result TBQ TBHQ

14. Morphology of cell

15. Morphology of cell Morphological change in apoptosis Fluorescence microscopy Complex morphological change in apoptosis Electron microscopy

16. 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

17. 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

18. Morphology of cells ResultFluorescence microscopy

19. Morphology of cells ResultElectron microscopy

20. Flow cytometry

21. Flow cytometry To investigate disruption of mitochondrial transmembrane potential

22. 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

23. 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.

24. Assay of caspase protease activities

25. 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.

26. Assay of caspase protease activities 2x106 cells/ml N-acetylcysteine medium GSH -TBHQ -TBQ Lysis buffer Cell lysate Enzyme activity

27. Assay of caspase protease activities Result

28. Assay of caspase protease activities Result Table1 Caspase activities induced by treatment of U937 cells with TBHQ and TBQ for 3 h

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

30. 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

31. 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

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

33. Release of cytochrome c from mitochondria to cytosol

34. 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

35. 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

36. 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.

37. Determination of reduced glutathione

38. Determination of reduced glutathione • Cellular glutathione which is an antioxidant contribute to defense against cell death. • DEVDase activity was inhibited by glutathione

39. Determination of reduced glutathione Method U937 cells TBHQ / TBQ PBS Cold perchlolic acid O-phthalaldehyde Cytofluor II

40. Determination of reduced glutathione result

41. Determination of cellular ATP

42. Determination of cellular ATP U937 Cells Glucose- containing medium Glucose-free medium TBHQ / TBQ Lysis buffer Supernatant Luminometer

43. Determination of cellular ATP Result

44. Discussion

45. 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.

46. Thank you for your attention