ALCOHOL AND ONCOGENESIS: The probable ALCOHOL AND ONCOGENESIS: The probable and the possibleand 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

A few facts about ethanol consumptionA 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

Talk structureTalk 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

Oxidation is Oxidation is removalremoval of a HYDRIDE (H of a HYDRIDE (H--) ) ionion

Oxidation of Primary AlcoholsOxidation of Primary Alcohols

Primary Primary AlcoholAlcohol

AldehydeAldehyde Carboxylic Carboxylic AcidAcid

R-R-CCH2-OHH2-OH R-R-CC-H-H R-R-CC-OH-OH

OOOO

Oxidation of Ethyl AlcoholOxidation of Ethyl Alcohol

CHCH33CHCH22OHOH CHCH33-C-O-C-O

OO

CHCH33 CH CH

OOEthanolEthanol AcetaldehydeAcetaldehyde AcetateAcetate

Ethanol MetabolismEthanol Metabolism

EthanolEthanol AcetaldehydeAcetaldehyde AcetateAcetate

Alcohol Alcohol dehydrogenasedehydrogenase

(ADH)(ADH)

Aldehyde Aldehyde dehydrogenasedehydrogenase

(ALDH)(ALDH)

MEOSMEOS

Oxidation of ethanol to acetaldehydeOxidation 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+

The Cytochrome P-450 complexThe 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

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.

CYP2E1 ReactionCYP2E1 Reaction

CYP2E1-generated 4-HNECYP2E1-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

Conversion of acetaldehyde to Conversion of acetaldehyde to acetateacetate

CHCH33-C-O-C-O

OO

CHCH33 CH CH

OO

+ H+ H22OOALDHALDH

NADNAD++ NADHNADH

Aldehyde dehydrogenase 2Aldehyde 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)

The Enzyme - ALDH2The Enzyme - ALDH2

Name:Name: Aldehyde dehydrogenase 2Aldehyde dehydrogenase 2

Location:Location: Mitochondrial matrixMitochondrial matrix

Kinetics:Kinetics: KKMM(acetaldehyde) = 0.2 (acetaldehyde) = 0.2 MM

KKMM(NAD(NAD++) = 70 ) = 70 MM

[NAD[NAD++]]mito mito = 6000 = 6000 MM

KKMM = = [S][S] atat V Vmaxmax

22

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

As [NADAs [NAD++]]mito mito >> K>> KMM(NAD(NAD++), an ), an enzyme-NADenzyme-NAD++ complex is always present complex is always present

i.e. the enzyme is saturated with NAD+i.e. the enzyme is saturated with NAD+

Reaction is pseudo first-order, rather Reaction is pseudo first-order, rather than second-orderthan second-order

• Enzyme is a Enzyme is a TETRAMER TETRAMER composed of 4 identical (composed of 4 identical () ) subunitssubunits

• MWMW = 54.422 kDa = 54.422 kDa

• Length = 517 amino acidsLength = 517 amino acids

ALDH2 StructureALDH2 Structure

• 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.

Ethanol Intolerance in AsiansEthanol Intolerance in Asians

• About 50% of Orientals (and many other About 50% of Orientals (and many other Asians) exhibit ethanol sensitivity.Asians) exhibit ethanol sensitivity.

• Manifests like an Antabuse (Disulfiram) Manifests like an Antabuse (Disulfiram) reaction: reaction:

facial flushingfacial flushingdysphoriadysphoriatachycardiatachycardianauseanauseahypotensionhypotension

WHY?WHY?

Because of a Because of a gg A mutation in A mutation in Exon 12 of ALDH2Exon 12 of ALDH2

Normal codon: gAA Glutamate (E) -ve

Mutant codon: AAA Lysine (K) +ve

i.e. E487K

Amino acid residue 487Amino acid residue 487

• E487 is an integral part of the subunit E487 is an integral part of the subunit interfaceinterface

• This site forms electrostatic and H bonds This site forms electrostatic and H bonds with Arg 475 in the adjacent subunitwith Arg 475 in the adjacent subunit

• Arg 475 normally stabilises the active Arg 475 normally stabilises the active site of ALDH2site of ALDH2

Mutation induced de-stabilisation increases Mutation induced de-stabilisation increases KKMM(NAD+) from 70 (NAD+) from 70 M to 7400 M to 7400 MM

R+ E- R+ K+

NB:NB: ALDH2 is a tetramerALDH2 is a tetramerThus, there is only Thus, there is only 1 chance in 161 chance in 16 of getting a of getting a normal enzyme (normal enzyme (44))

A Dominant MutationA Dominant Mutation

IsozymeIsozyme 44 33 2222 33 44

ProportionProportion 11//1616 11//4 4

33//8 8 11//44

11//16 16

• Frequency of mutation probably Frequency of mutation probably represents a represents a FOUNDER EFFECTFOUNDER EFFECT

• The mutation occurred 2000-3000 years The mutation occurred 2000-3000 years ago in a Han Chinese populationago in a Han Chinese population

i.e. The i.e. The E487K E487K mutation was present in a mutation was present in a small ancestral population that gave rise small ancestral population that gave rise to present day Asiansto present day Asians

• Is present in some 560 million Asians (8% Is present in some 560 million Asians (8% of the human population)of the human population)

Reactivity and toxicity of acetaldehydeReactivity 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

CrotonaldehydeCrotonaldehyde

• CH3 CH CH CH O

Generation of acetateGeneration 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!

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 2015. 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.

Major ALDH2 referenceMajor ALDH2 reference

• TARGETING ALDEHYDE DEHYDROGENASE 2: NEW THERAPEUTIC OPPORTUNITIES

Che-Hong Chen et al. Physiol. Rev. 2014; 94:1- 34.