1. Toxicity of Acetaldehyde with Oxygen Radicals Heather Bolstad Mentor: Joseph S. Beckman, Ph.D. August 28, 2003

2. A Model for Alcohol Toxicity  Ethanol (CH 3 CH 2 OH) is oxidized to acetaldehyde (CH 3 CHO) in the liver by the enzyme alcohol dehydrogenase  Oxygen radicals produced in cells via electron-transfer processes and respiration  Formed from the reduction of molecular oxygen  Highly reactive due to an unpaired electron, represented by a ˙ Goodsell, David S. Protein Data Bank Molecule of the Month: Alcohol Dehydrogenase.”

3. Motivation for the Assay  Studies concerning the bacterial toxicity of extracellular oxygen radicals have yielded conflicting results  Rosen and Klebanoff (1979, 1981) found that the enzyme xanthine oxidase (XO) generated a substance toxic to Staphylococcus aureus with 10 mM acetaldehyde as the substrate “Crystal Structure of Xanthine Oxidase from Bovine Milk.” Protein Data Bank. 4 Aug. 2000. http://www.rcsb.org/pdb/cgi/explore.cgi?job=graphics;pdbId=1FIQ &bio=1&opt=show&size=250. http://www.rcsb.org/pdb/cgi/explore.cgi?job=graphics;pdbId=1FIQ &bio=1&opt=show&size=250

4. Xanthine Oxidase (XO) Mechanism XO XO Xanthine Hypoxanthine Uric acid O 2 O 2 ˙ - O 2 O 2 ˙ - H 2 O 2 H 2 O 2 Metal Catalyst Metal Catalyst 2 HO˙

5. Alternative XO Mechanism acetaldehyde XO acetic acid O 2 O 2 ˙ - H2O2H2O2 1-hydroperoxyethanol peracetic acid oxidation bacteria killed!

6. Acetaldehyde and H 2 O 2 Reactions  Unclear whether toxicity was due to the products of XO (O 2 ˙ -, H 2 O 2 ) or from the acetaldehyde/H 2 O 2 adduct: OH oxidation O CH 3 CHO + H 2 O 2 CH 3 -C-O-OH CH 3 -C-O-OH H acetaldehyde 1-hydroperoxyethanol peracetic acid

7. Dr. Beckman’s Research (1984)  Acetaldehyde plus H 2 O 2 was bactericidal (P. fluorescens) Acetaldehyde, H 2 O 2 not toxic individually Toxicity inhibited by catalase or SOD  XO reaction (P. fluorescens and S. aureus) Toxic with 10 mM acetaldehyde  Toxicity inhibited by SOD or catalase Not toxic with xanthine  Superoxide and hydroxyl radical formation detected so they must not be toxic Toxic with 1 mM acetaldehyde plus xanthine  Not toxic when substrates were tested separately  Conclusions: Acetaldehyde and H 2 O 2 involved in toxicity

8. The Goal:  To determine the toxicity of acetaldehyde with the oxygen radicals 1. superoxide, O 2 ˙ - 2. hydrogen peroxide, H 2 O 2 3. hydroxyl radical, HO˙

9. Hypothesis  Acetaldehyde forms toxic adducts with superoxide radical, hydrogen peroxide, and hydroxyl radical  The number of E. coli colonies that survive will be used to determine the toxicity of the adduct

10. Procedure Early log-phase E. coli washed 2X and resuspended in buffer Titer of ~10 7 cells/mL made in buffer Cells, DTPA, and test reagents incubated in buffer at room temp. 1:10 1:10 1:10 Microdrops onto LB agar plates Incubated overnight at 37  C Colonies counted/averaged; % survival determined

14. Modifications to Assay  Hydrogen peroxide solutions were checked with a UV spectrophotometer  New cell line, E. coli B, substituted for E. coli JM 109  MgSO 4 added to stabilize bacterial membranes  Used pH 5.0 buffer instead of pH 7.4

16. Toxicity of the Xanthine Oxidase Reaction

17. Future Experiments  Test P. fluorescens and compare to E. coli  Test pure peracetic acid on both species  Modify washing procedure to prevent growth of bacteria throughout assays

18. Acknowledgements Howard Hughes Medical Institute Joseph S. Beckman, Ph.D. Linus Pauling Institute Patrick Reardon Kevin Ahern, Ph.D. Environmental Health Sciences Center Department of Biochemistry and Biophysics