Mikko Salaspuro, prof., M.D., Ph.D., Research Unit on Acetaldehyde and Cancer

University of Helsinki, Helsinki, Finland COI: Board member and stock owner of Biohit Oyj., Helsinki

ACETALDEHYDE THE MOST COMMON HUMAN CARCINOGEN

mikko.salaspuro@helsinki.fi

ESBRA 2015 Charles S Lieber

Memorial Lecture

WHAT

WHY

HOW

1931 - 2009

PRINCIPLES OF SCIENCE

CONTENTS

1. What is acetaldehyde (AA)

2. Why AA is the most common human carcinogen

3. How upper digestive tract is exposed to AA

4. ALDH2-deficiency as a unique human model for local AA exposure and cancer

5. Tobacco smoking and AA

6. Inconsistent opinions of authorities on the carcinogenicity of AA

7. Stomach cancer and AA

8. Prevention of AA related cancers

ACETALDEHYDE

Easily soluble to water and lipids

Free aldehyde group

Passes cell

mem- branes

Carcinogenic DNA adducts in oral mucosa in man

Carcinogenic to animals

Acetaldehyde – most common human carcinogen

Aroma ?

Either present or formed by oxidation

from ethanol

Last metabolite of alcohol fermentation

from glucose to ethanol

First metabolite of alcohol oxidation from ethanol to

acetate

Acetaldehyde – most common human carcinogen

Particularly high concentrations in some alcoholic beverages

Chinese spirits up to

26.000µM

Mexican spirits up to

16.000µM

Grappa, Calvados, Sherry

400 to 12.000 µM

Zero or low concentrations in some other alcoholic beverages

Zero acetaldehyde

in well distilled spirits

In beers may range from 0 to 1400µM

Rather low levels in most wines, since

free acetaldehyde is bound to sulphites

Ethanol and/or AA of Japanese fermented foods

FOOD ETHANOL VOL. %

ACETALDEHYDE µM

Soya sauce 8,75 296

Miso 5,55 233

Nakazuke (Fermented rice brand)

1,20 1296

Narazuke (Vegetables pickled in sake lees)

16,34 278

Umeboshi (Salted apricot) 3,94 96

Kimuchi (Korean pickles) 0,11 1384

Salted fish guts 2,93 211

Traditional sake 15,07 146

Within 12 minutes cooking of rice with ethanol only 50 – 40% of ethanol is evaporated

RICE

Effect of cooking on ethanol and AA of Japanese food

MISO-MARINATED FISH 1 3/4 tea spoon miso paste 1 tea spoon sake, mirin and soya

sauce 3 tea spoons sugar Marinated for 16 hours and

cooked in oven

SUKIYAKI HOT POT 0.5dl mirin (EtOH 0.37 %) 0.5dl sake (EtOH 15.5 %) 0.4dl soya sauce (EtOH 5.73%) 2 spoons sugar Cooked in hot pot for ten minutes

Ethanol and/or AA in Japanese food after cooking

FOOD ETHANOL VOL. %

ACETALDEHYDE µM

Miso-marinated fish - Fish - Soba Noodels - Marinade - Noodel soup

1.06 0.54 2.03 3.55

79 39

121 131

Sukiyaki-hot pot dish - Marinade - Vegetables - Meat - Tofu

0.70 0.55 0.49 0.28

71 51 55 30

It is well known that ethanol is metabolized in the liver so effectively that no acetaldehyde is released to the blood

Less well is known that a minor amount of ethanol is metabolized locally to acetaldehyde in the mouth and stomach

Acetaldehyde exposure is localized to the upper G-I tract

Salivary glands, oral microbes and mucosal cells are able to metabolize alcohol to acetaldehyde

However, the ability microbes and mucosa to eliminate acetaldehyde is low

Therefore, in the presence of any alcohol in the mouth or stomach carcinogenic levels of acetaldehyde accumulate in the saliva and gastric juice

Microbes

Mucosal cells

Salivary glands

Acute effect of alcohol sipping on salivary acetaldehyde

Linderborg et al. Food Chem Tox 2011;49:2103-6

5ml of alcoholic beverage in mouth for 5 seconds Ethanol stays in

saliva for up to 15 minutes

High acetaldehyde concentration of the beverage results in 2 minute’s additional exposure of the mouth to acetaldehyde

1 drink of alcohol daily increases

significantly risk for oral cancer

Mutagenic level

After a moderate dose (0.5g/kg) of alcohol: - normal - with chlorhexidine

rinsing Ethanol is distributed

to saliva from the blood and stays in saliva for up to 4 hours 0 20 60 100 140 180 220 260

0

10

20

30

40

µ MN o rm a l

C h lo rh e x id in e +

M in u tes

Homann et al. Carcinogenesis 1997

Long-term effect of alcohol drinking on salivary acetaldehyde

Mutagenic level

CARCINOGENICITY OF ACETALDEHYDE IS BASED ON A UNIQUE HUMAN CANCER MODEL CAUSED BY GENE ERROR

Point mutation in ALDH2-gene Results in deficient ALDH2-enzyme

Most common one point mutation related health risk in man Incidence 1:13 That of familiar hypercholesterolemia is 1:500

Equal model - randomized by nature - is not available for any other of group 1 human carcinogens (n = 113) Most importantly, possible confounding factors can

be assumed to be evenly distributed among ALDH2-actives and -deficients

Point mutation in ALDH2 gene took place in South China over 2000 years ago

Results in deficient ALDH2 enzyme in all cells Affects about 600 million subjects with East

Asian descent In ALDH2-deficient individuals alcohol drinking

results in markedly elevated local acetaldehyde exposure of digestive tract mucosa via saliva and gastric juice Unique model for local AA exposure

Ethanol ACETALDEHYDE Acetate

ALDH2

Saliva Gastric juice

Effect of a moderate dose (0.5g/kg) ethanol on salivary and gastric juice acetaldehyde levels in ALDH2-actives

compared to ALDH2-deficients

Väkeväinen et al. AlcClinExpRes 2000 Maejima et al PLOS ONE 2015

More alcohol-longer exposure

Mutagenic level

Mutagenic level

In lines with biochemical findings ALDH2-deficient alcohol drinkers have markedly higher upper digestive tract cancer risk than in those with the active ALDH enzyme

RR

Cancer risk in ALDH2-deficient heavy drinkers compared to

ALDH2-actives

Confirmed in 10s of studies and meta-analyses

Yokoyama et al. Carcinogenesis 1998

High active ADH1C*1

Rate of ethanol oxidation 2.5-fold

Increased salivary acetaldehyde levels after alcohol intake

Significantly increased incidence of upper aerodigestive tract cancers among heavy drinkers

Visapää et al. Gut 2004;53:871-6

Based on gene-epidemiological and gene-biochemical studies IARC

concluded in 2009 that ACETALDEHYDE associating with alcohol is

GROUP 1 human carcinogen

Concerns oral-, pharyngeal- and esophageal

cancers

Further evidence for the local carcinogenicity of AA in man

One oral dose of alcohol results in dose dependent increase in carcinogenic AA-DNA adducts (N2-ethylidene-dGuo) in human oral mucosa

One alcohol dose is known to result in 20 to 200µM levels of AA in the saliva

0

5000

10000

15000

20000

25000

0.3 0.5 0.6

Fmo

l/µ

mo

l dG

uo

Dose of alcohol g/kg

AA-DNA-adducts at 2hrs from alcohol drinking

Base line

(Balbo et al. Cancer Epid Biomark Prev 2012;21:601-8)

Drinking of 5% ethanol or water for 8 weeks

Isolation of esophageal DNA

12-fold increase in mutagenic N2-ethylidene-DGuo

Major mutagenic AA-DNA adduct

0

2

4

6

8

10

12

Water Ethanol

Ad

du

ct le

vel/

10

7 b

ase

s

AA-DNA adducts

Control ALDH2-

(Yakawa et al. Am J Cancer Res 2014;4:279-284)

Further evidence for the local carcinogenicity of AA from ALDH2-knock-out mice

Tobacco smoking and acetaldehyde

Tobacco smoking

Independent risk factor for oral, pharyngeal, esophageal and stomach cancer

Synergistic effect with alcohol on upper G-I tract cancer risk

Acetaldehyde

Most abundant carcinogenic compound of tobacco smoke

Dissolves easily into the saliva

Is distributed via saliva to the mucosa of the whole upper digestive tract

Synergistic effect of alcohol and tobacco on the risk for oesophageal cancer (Tuyns et al. Bull du Cancer 1977)

0-40

40-80

80-120

>120

0

50

100

150

0…10 10…30 > 30

RR

Tuyns et al. Bull du Cancer 1977

Alcohol

g/day

Cigarettes/day

Smoking and alcohol have a synergistic effect on

salivary acetaldehyde (Tobacco smoking modifies oral flora to

produce more AA from ethanol)

Smoking: about 5 min.

Salaspuro V et al., Int J Cancer 2004;111:480-3

Alcohol 0.8g/kg

AUC: 7-fold in smokers as compared

to non-smokers p < 0.001

Mutagenic level

Inconsistent opinions of authorities

IARC/WHO (2009) AA associated with consumption of alcoholic

beverages is carcinogenic to humans (Group 1)

Joint FAO/WHO Expert Committee on Food Additives (JEFCA) in 1998 AA is a GRAS product (Generally Regarded as Safe) Is not based on present scientific evidence

Scientific Committee on Consumer Safety (SCCS/EC, 2012) Maximum concentration for acetaldehyde in the final

finished cosmetic product 5mg/l (114µM) Not be intentionally used in mouth-washing products

AA

Stomach cancer - third leading cause of cancer death in both sexes worldwide

ACETALDEHYDE (AA) IS A COMMON DENOMINATOR

Helicobacter pylori

Atrophic gastritis

ALDH2-deficiency

Smoking

Heavy drinking

Fermented food

RISK CONDITIONS RISK FACTORS

Atrophic gastritis acid free stomach

0

5

10

15

20

25

30

35

40

45

30 min. 60 min.

Controls Achlorhydrics

+ ethanol 15%

Oral microbes

Colonize acid free stomach

Produce AA locally from any alcohol present in beverages or food

Also glucose may serve as a source for AA

Gastric juice AA, µM

1 0 2 0 4 0 6 0 8 01 0 0

1 2 01 4 0

1 6 01 8 0

2 0 02 2 0

2 4 0

0

2 0

4 0

6 0

µ M

M in u te s

Gastric juice AA in relation to g.j. ethanol in patients with atrophic gastritis (n=7)

Hellström et al. DDW 2014

Alcohol (15 vol.%, 0.3g/kg

Gastric juice ethanol levels under the official limit for

alcoholic beverages (2.0 – 2.8 vol. %)

Gastric juice AA in relation to g.j. ethanol in PPI treated ALDH2-deficient patients (n=10)

Alcohol (15 vol.%, 0.5g/kg

0 3 0 6 0 9 0 1 2 0

0

2 0

4 0

6 0

8 0

µ M

M in u te s Maejima et al. 2015, PLOS ONE

Gastric juice ethanol levels under the official limit for

alcoholic beverages (at 120min. 1.3‰)

CONCLUSIONS with regard to stomach cancer

1. Many commercially available food stuffs contain marked levels of ethanol and acetaldehyde

2. Ethanol and acetaldehyde derived from these products expose upper digest tract mucosa to mutagenic concentrations of acetaldehyde

3. The fact that ethanol and acetaldehyde intake via widely used foodstuffs and beverages is not systematically recorded causes an obvious, but yet unrecognized, confounder and bias in cancer epidemiology of the upper digestive tract.

Basics for cancer prevention 1

Identification of spesific carcinogenic agent

AA = Group 1 human carcinogen comparable to asbestos, benzene, formaldehyde and radon

ALDH2-deficiency provides a unique human cancer model for local acetaldehyde exposure

Recognition of the wide presence of carcinogenic agent (AA) in our daily environment

Acetaldehyde probably is the most common and prevalent human carcinogen

Basics for cancer prevention 2

Knowledge of mechanisms regulating local AA concentration in the upper digestive tract

Great individual variation in salivary AA after alcohol drinking is due to individual microbial flora

Good oral hygiene results in lower AA exposure

Risk factors enhancing AA exposure: ALDH2-deficiency, high active ADH, atrophic gastritis, H. pylori infection, use of drugs inhibiting secretion of gastric acid (PPIs, H2-blockers)

Drinking habits: Higher ethanol ► higher AA ►longer exposure time

How to minimize acetaldehyde exposure

Prefer beverages with low free acetaldehyde

Avoid beverages and food containing low levels of ethanol

Result in marked local production of AA

Avoid beverages and food containing high levels of free acetaldehyde

AA AROMA

How to minimize acetaldehyde exposure

L-CYSTEINE in the minimization of AA exposure

Normal and safe amino acid that binds covalently and

non-enzymatically to acetaldehyde and forms

Inactive 2-methyltiazolidine-4-carboxylicacid (MTCA)

+

Slow release l-cysteine (200mg) eliminates effectively AA from gastric juice of ALDH2-deficient PPI-users after

intragastric dose of alcohol (0.5g/kg)

Maejima et al. 2015, PLOS ONE

Mutagenic level

Slow release L-cysteine (200mg) effectively eliminates AA from gastric juice of patients with atrophic gastritis after

intragastric installation of alcohol (0.3g/kg)

68 % decrease P < 0.0001

Hellström et al. DDW 2014

Mutagenic level

L-cysteine and MTCA levels of gastric juice after slow release L-cysteine (200mg) and intragastric installation of

alcohol (0.3g/kg)

L-cysteine and MTCA persist in the stomach for up to 3 hours

Min.

Hellström et al. DDW 2014

FINAL CONCLUSIONS

1. Acetaldehyde (AA) associated with alcohol is the most prevalent human carcinogen (Group 1)

2. Local carcinogenicity of AA in the upper digestive tract is based on a unique human model

3. There is no evidence that easily water soluble AA derived from tobacco or low ethanol/high AA beverages and food is less carcinogenic than AA associated with use of alcoholic beverages

4. AA is a cumulative carcinogen

5. AA exposure can be markedly decreased both at population and individual level

STRONG EVIDENCE

MISSING ACTIONS

CARCINOGENICITY OF ACETALDEHYDE

Acknowledgements

• Research Unit on Acetaldehyde and Cancer, University of Helsinki – Katja Salmela, Risto Roine, Nils Homann, Kalle Jokelainen, Jyrki Tillonen,

Jukka-Pekka Visapää, Ville Salaspuro, Tiina Koivisto, Tatiyna Nosova, Klas Linderborg, Johanna Uittamo (Kurkivuori), Pertti Kaihovaara, Satu Väkeväinen, Mikko Nieminen, Andreas Helminen, Kalle Nummi

• Department of pharmacy, University of Helsinki – Martti Marvola, Alma Kartal, Tuuli Marvola

• Department of Gastroenterology, Helsinki University Central Hospital – Martti Färkkilä, Hannu Nuutinen

• Salem Medical Center, Heidelberg, Germany – Helmuth Seitz

• National Cancer Institute, NIH – Neal Freedman, Christian Abnet, Sandy Dawsey

• Division of Gastroenterology, Tohoku University, Sendai, Japan – Ryehui Maejima, Katsunori Iijima, Tooru Shimosegawa

• Department of Medical Sciences, Uppsala University, Sweden – Per Hellström