toxicity of acetaldehyde with oxygen radicals heather bolstad mentor: joseph s. beckman, ph.d....
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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)
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