1. ALCOHOL AND ONCOGENESIS: The probable and the possible
The global burden of alcohol is 3.8% of all deaths (men 6.3%, women 1.1%).
The proportion of cancers attributable to alcohol worldwide is 3.6%, and the proportion of cancer deaths is 3.5 to 5.0%.
The importance of alcohol as a carcinogen is generally underestimated at the public level

2. A few facts about ethanol consumption
Per-capita consumption is highest in eastern Europe and the Russian Federation
Over the past 40 years, consumption has increased 5 times in China
The effects of drinking and smoking seem to be multiplicative
The proportion of cancers attributable to ethanol consumption is probably higher than previously estimated

3. Talk structure The oxidation of ethanol—pathways
Alcohol dehydrogenase (ADH) and CYP2E1
Aldehyde dehydrogenase (ALDH)
Acetaldehyde—the main villain in the story
Crotonaldehyde and 4-HNE---reactions with DNA
Acetate as food for tumours

4. Oxidation of Primary Alcohols
Carboxylic Acid
Aldehyde
R-CH2-OH
R-C-H
R-C-OH O O
Oxidation is removal of a HYDRIDE (H-) ion

5. Oxidation of Ethyl Alcohol
CH3CH2OH
CH3 CH O
CH3-C-O O
Ethanol
Acetaldehyde
Acetate

6. Ethanol Metabolism Alcohol dehydrogenase (ADH) Aldehyde dehydrogenase
(ALDH)
Acetaldehyde
Ethanol
Acetate
MEOS

7. Oxidation of ethanol to acetaldehyde
There is a multitude of ADHs in the cytosol of the cell
They vary in both their affinity for ethanol and their rate of metabolism (Vmax)
In Caucasians it is unlikely that ADH enzyme diversity is relevant to ethanol-induced pathology
The reaction: Ethanol + NAD+ + H2O
Acetaldehyde + NADH + H+ O

8. The Cytochrome P-450 complex
This membrane-associated enzyme superfamily is essentially a cellular detox unit
Used to be called the MEOS (microsomal ethanol oxidizing system)
Consists of 57 different proteins
The one we are interested in is called Cytochrome P-450 2E1 (CYP2E1)
This enzyme efficiently metabolizes ethanol and other compounds, including lipids and components of cigarette smoke

9. The Cytochrome P-450 system is INDUCIBLE.
ie. Exposure to a particular compound induces the enzyme(s) that metabolise that substrate.
In the case of ethanol, the major inducible cytochrome P-450 component is Cytochrome P-450 2E1 (CYP2E1).
Induction occurs in response to surprisingly modest amounts of ethanol.
Upon removal of the substrate, enzyme levels decline rapidly.

10. CYP2E1 Reaction

11. CYP2E1-generated 4-HNE
CYP2E1 converts products of lipid (fat) oxidation to 4-hydroxy-2-nonenal (4-HNE)
This molecule reacts with DNA to form mutagenic etheno-DNA adducts

12. Conversion of acetaldehyde to acetate
ALDH
CH3 CH O
CH3-C-O O
+ H2O
NAD+
NADH

13. Aldehyde dehydrogenase 2
This enzyme is one of 19 aldehyde dehydrogenases in the human genome
ALDH2 is responsible for almost all acetaldehyde oxidation in the cell (we know this because of ethanol intolerance in Asians with a non-functional form of the enzyme)
It is located in the mitochondrial matrix and is found in nearly all cell types (not just in hepatocytes)

14. The Enzyme - ALDH2 Name: Aldehyde dehydrogenase 2
Location: Mitochondrial matrix
Kinetics: KM(acetaldehyde) = 0.2 mM
KM(NAD+) = 70 mM
[NAD+]mito = 6000 mM

15. KM = [S] at Vmax 2
The Km is not only a measure of the affinity of an enzyme for it’s substrate(s), but is the substrate concentration at half-maximal velocity

16. As [NAD+]mito >> KM(NAD+), an
enzyme-NAD+ complex is always present
i.e. the enzyme is saturated with NAD+
Reaction is pseudo first-order, rather than second-order

17. ALDH2 Structure
Enzyme is a TETRAMER composed of 4 identical (a) subunits
MWa = kDa
Length = 517 amino acids a a a a a

18. 4-HNE is a substrate for ALDH2, with a Km of 14 µM.
4-HNE also inhibits the enzyme by forming a Michael adduct or Schiff base with cysteine 302 at the catalytic site, with a Ki of 0.5 µM (at a steady-state conc. of 0.50 µM 4-HNE, acetaldehyde oxidation would be reduced by 50%)
One of the 19 ALDHs, cytosolic ALDH1A1, oxidizes 4-HNE, with a Km of ~18 µM.

19. Ethanol Intolerance in Asians
About 50% of Orientals (and many other Asians) exhibit ethanol sensitivity.
Manifests like an Antabuse (Disulfiram) reaction:
facial flushing
dysphoria
tachycardia
nausea
hypotension

20. Because of a g A mutation in
WHY?
Because of a g A mutation in
Exon 12 of ALDH2
Normal codon: gAA Glutamate (E) -ve
Mutant codon: AAA Lysine (K) +ve
i.e. E487K

21. Amino acid residue 487
E487 is an integral part of the subunit interface
This site forms electrostatic and H bonds with Arg 475 in the adjacent subunit
Arg 475 normally stabilises the active site of ALDH2

22. R+ E- R+ K+
Mutation induced de-stabilisation increases KM(NAD+) from 70 mM to 7400 mM

23. A Dominant Mutation NB: ALDH2 is a tetramer
Thus, there is only 1 chance in 16 of getting a normal enzyme (a4)
Isozyme b4 b3a b2a2 ba3 a4
Proportion 1/16 1/ / /4 1/16

24. Frequency of mutation probably represents a FOUNDER EFFECT
The mutation occurred years ago in a Han Chinese population
i.e. The E487K mutation was present in a small ancestral population that gave rise to present day Asians
Is present in some 560 million Asians (8% of the human population)

25. Reactivity and toxicity of acetaldehyde
Acetaldehyde reacts with primary amines (-NH2 groups) to form crotonaldehyde, which reacts with deoxycytidine and deoxyadenosine bases in DNA.
These modified bases are mutagenic, resulting in misrepair and mispriming in the DNA molecule
The end result is the introduction into the DNA of stable mutational changes

26. Crotonaldehyde
CH3 CH CH CH O

27. ATP + Acetate + Coenzyme A PPi + AMP + Acetyl Coenzyme A
Generation of acetate
Acetate derived by oxidation of acetaldehyde diffuses out of the liver and is converted to Acetyl-CoA, mainly in muscle and fat tissue.
ATP + Acetate + Coenzyme A PPi
+ AMP + Acetyl Coenzyme A
This seems innocuous, but it may not be!

28. The enzyme involved is called Acetyl CoA Synthetase 2 and it has very recently been shown to be up-regulated in some cancer tissues
Cancer Cell. Jan Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.
LM5, Smethurst E4, Mason S1, Blyth K1, McGarry L1, James D1, Shanks E1, Kalna G1, Saunders RE2, Jiang M2, Howell M2, Lassailly F2, Thin MZ2, Spencer-Dene B2, Stamp G2, van den Broek NJ1, Mackay G1, Bulusu V7, Kamphorst JJ7, Tardito S1, Strachan D1, Harris AL3, Aboagye EO6, Critchlow SE5, Wakelam MJ4, Schulze A2, Gottlieb E8.

29. Major ALDH2 reference
TARGETING ALDEHYDE DEHYDROGENASE 2: NEW THERAPEUTIC OPPORTUNITIES
Che-Hong Chen et al. Physiol. Rev. 2014; 94: