Toxicity of Acetaldehyde with Oxygen Radicals

Heather Bolstad

Mentor: Joseph S. Beckman, Ph.D.

August 28, 2003

A Model for Alcohol Toxicity Ethanol (CH3CH2OH) is oxidized to

acetaldehyde (CH3CHO) 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.” <www.rcsb.org/pdb/molecules/pdb13_1.html>

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.

Xanthine Oxidase (XO) Mechanism XO XOXanthine Hypoxanthine Uric acid

O2 O2˙- O2 O2˙-

H2O2 H2O2

Metal Catalyst Metal Catalyst

2 HO˙ 2 HO˙

Alternative XO Mechanism

acetaldehydeXO

acetic acid

O2 O2˙-

H2O2

1-hydroperoxyethanol

peracetic acidoxidation bacteria killed!

Acetaldehyde and H2O2 Reactions Unclear whether toxicity was due to the products of

XO (O2˙-, H2O2) or from the acetaldehyde/H2O2 adduct:

OH oxidation O

CH3CHO + H2O2 CH3-C-O-OH CH3-C-O-OH

H

acetaldehyde 1-hydroperoxyethanol peracetic acid

Dr. Beckman’s Research (1984) Acetaldehyde plus H2O2 was bactericidal (P. fluorescens)

Acetaldehyde, H2O2 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 H2O2 involved in toxicity

The Goal:

To determine the toxicity of acetaldehyde with the

oxygen radicals

1. superoxide, O2˙-

2. hydrogen peroxide, H2O2

3. hydroxyl radical, HO˙

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

ProcedureEarly log-phase E. coli washed 2X and resuspended in buffer

Titer of ~107 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

1

10

100

0 2 4 6 8 10

Acetaldehyde Dose-Response Curve: JM 109 E. coli

Incubation time: 60 minutes

% S

urv

ival

Acetaldehyde (mM)

0 mM and 0.1 mM acetaldehyde

0.01

0.1

1

10

100

0 500 1000 1500 2000

Hydrogen Peroxide Dose-Response Curve: JM 109 E. coli

Incubation time: 60 minutes%

Su

rviv

al

Hydrogen peroxide (uM)

0.01

0.1

1

10

100

0 50 100 150 200

Bactericidal Activity of 50 uM Acetaldehyde with Hydrogen Peroxide: JM 109 E. coli

Incubation time: 60 minutes

50 uM Acetaldehyde with Hydrogen PeroxideHydrogen peroxide dose response

% s

urv

ival

Hydrogen peroxide (uM)

Modifications to Assay Hydrogen peroxide solutions were checked with

a UV spectrophotometer

New cell line, E. coli B, substituted for E. coli

JM 109

MgSO4 added to stabilize bacterial membranes

Used pH 5.0 buffer instead of pH 7.4

0.01

0.1

1

10

100

-50 0 50 100 150 200 250

Hydrogen Peroxide Dose Response: JM 109 vs. B E. coliIncubation time: 60 minutes

% survival of JM 109

% survival of B %

su

rviv

al

Hydrogen peroxide (uM)

Toxicity of the Xanthine Oxidase Reaction

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

AcknowledgementsHoward 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