Download Toxicity of Acetaldehyde with Oxygen Radicals

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
XO
Xanthine
Hypoxanthine
O2
O 2 ˙-
Uric acid
O2
O 2 ˙-
H2O2
H2O 2
Metal Catalyst
Metal Catalyst
2 HO˙
2 HO˙
Alternative XO Mechanism
XO
acetaldehyde
acetic acid
O2
1-hydroperoxyethanol
oxidation
˙
O2 -
H2O2
peracetic acid
bacteria killed!
Acetaldehyde and H2O2 Reactions

Unclear whether toxicity was due to the products of
XO (O2˙-, H2O2) or from the acetaldehyde/H2O2
adduct:
CH3CHO + H2O2
acetaldehyde
OH
CH3-C-O-OH
H
oxidation
1-hydroperoxyethanol
O
CH3-C-O-OH
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


Not toxic with xanthine


Superoxide and hydroxyl radical formation detected so they must
not be toxic
Toxic with 1 mM acetaldehyde plus xanthine


Toxicity inhibited by SOD or catalase
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
Procedure
Early 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
Acetaldehyde Dose-Response Curve:
JM 109 E. coli
Incubation time: 60 minutes
% Survival
100
0 mM and 0.1 mM acetaldehyde
10
1
0
2
4
6
8
Acetaldehyde (mM)
10
Hydrogen Peroxide Dose-Response Curve:
JM 109 E. coli
Incubation time: 60 minutes
100
% Survival
10
1
0.1
0.01
0
500
1000
1500
Hydrogen peroxide (uM)
2000
Bactericidal Activity of 50 uM Acetaldehyde
with Hydrogen Peroxide: JM 109 E. coli
Incubation time: 60 minutes
50 uM Acetaldehyde with Hydrogen Peroxide
Hydrogen peroxide dose response
% survival
100
10
1
0.1
0.01
0
50
100
Hydrogen peroxide (uM)
150
200
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
Hydrogen Peroxide Dose Response:
JM 109 vs. B E. coli
Incubation time: 60 minutes
% survival of JM 109
100
% survival of B
% survival
10
1
0.1
0.01
-50
0
50
100
150
200
Hydrogen peroxide (uM)
250
Toxicity of the Xanthine Oxidase Reaction
Percent survival
Experiment
P. fluorescens
S. aureus
E. coli B
Xanthine
100 (0.1 mM)
100 (0.1 mM)
99.8 (0.2 mM)
Acetaldehyde
0.05 (10 mM)
0.3 (10 mM)
95.5 (10 mM)
0.08 (1 mM/0.1 uM)
<0.01 (1 mM/0.1 uM)
99.0 (10 mM/1 mM)
Acetaldehyde + Xanthine
P. fluorescens and S. aureus exposure for 60 min.; E. coli B exposure for 20 min. (number is average over time course).
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
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