Low Dose Effects of the Dietary Carcinogen Acrylamide (AA)

Gerhard Eisenbrand

3rd International Summit on Toxicology and Applied Pharmacology

October 20-22, 2014, Chicago,USA

Senior Research Professor-retired

University of Kaiserslautern

Department of Chemistry

Division of Food Chemistry and Toxicology

Kaiserslautern, Germany

Heat-treatment of food

Non enzymatic browning � “ Maillard-reaction chemistry“

reaction of reducing sugars with amino acids

First described 1912 by the French chemist

Louis Camille Maillard

heating alanine and glucose �carbon dioxide+water and brown colour

2

Heat Processing: formation of bioactive compounds in food by „Maillard-chemistry“

colorants

flavourtaste

modified proteins

food-borne toxicants:Acrylamide,Furan,Nitrosamines

Heterocyclic aromatic amines

Acrolein

antioxidants

3

NH2

NH2

O

COOH NH2

O

COOH

NH

R

OH

HO

R

H

H2O

Reducing sugar

Asparagine

Tareke et al 2002; Rosen and Hellenas, 2002; Zyzak et al., 2003

AA: Formation in foods by thermal treatment

NH2

O

COOH

NCH

R

NH2

O HNCH

R

NH2

O HNCH

R

NH2

O

NH

R

H

NH2

O NH2

NH2

O

R

O

H

CO2

++

NH3

Schiff base

Acrylamide Acrylamide3-Aminopropionamide

H2O

4

Exposure to Acrylamide (AA)

� Highly variable ���� Individual consumption habits / contents of individual food groups

� Dietary sources: potato fried products (up to 50%), soft / crisp bread, biscuits, crackers other products based on cereals / potatoes,coffee / coffee substitutes

� No noteworthy exposure to AA from environmental sources (except tobacco smoke)

� Endogenous metabolic formation of AA ? scarcely studied, suggestive evidence in rats and humans

���� Estimated daily uptake (µg/kg b.w./day ; EFSA, 2014)

Infants, toddlers,children: 0.5-1.9 (average);1.4-3.4 (95th%ile);

Adolescents, adults, elderly: 0.3-0.9 (average);0.6-2.0 (95th%ile);

5

AA: Hazard Characterisation

(EFSA Contam Panel, draft opinion on AA in foods, 2014)

Critical endpoints for toxicity (animal studies):

Neurotoxicity: peripheral/central axono-/neuropathy in several species,

including humans

Benchmark dose ����BMDL10 : 0,43 mg/kg b.w.;

Carcinogenicity: Harderian gland tumors in mice

Benchmark dose ����BMDL10 : 0,17 mg/kg b.w.;

AA is considered a genotoxic carcinogen, acting through metabolic

conversion to epoxypropaneamide (Glycidamide,GA), a DNA damaging

(genotoxic) mutagen

6

.

Benchmark dose (BMD) : dose related to a defined (benchmark) response ( eg 10% = BMD10)

���� Lower Confidence Limit (BMDL10)

MOE : benchmark dose level divided by human exposure

MOE > 10.000 : „of low concern from a public health point of view…

…“low priority for risk management“

Benchmark dose and Margin of Exposure (MoE)for Genotoxic Carcinogens in Food

[O‘Brien et al., Food Chem Tox 44 (2006) 1613-1635; Constable A. and Barlow S, ILSI Europe Report Series 2009] 7

Dose-response curve showing how the BMDL is derived. BMR, benchmark response; BMD10, dose calculated

to cause a 10 % increase in the background incidence of tumors; BMDL10, lower confidence limit of the BMD10

AA: risk characterisation

(EfSA Contam Panel, Draft opinion on AA in foods (2014)

Human dietary exposure : potato fried products (up to 50%), soft/ crisp bread,

biscuits, crackers, other products based on cereals / potatoes,coffee/ coffee substitutes

Infants,toddlers,children (µg/kg b.w./day) : 0.5-1.9 (average); 1.4-3.4 (95th%ile);

Adolescents, adults,elderly : 0.3-0.9 (average); 0.6-2.0 (95th%ile);

Margins of exposure (MOEs)

Neurotoxicity : dietary exposure ���� no concern (thresholded ���� NOEL >100)

Neoplastic effects : dietary exposure ���� of concern

MOE mean exposure : 567 (min. lower bound) to 89 (max.upper bound)

MOE 95th%ile exposure : 283 (min. lower bound) to 50 ( max.upper bound)

8

Cancer is considered the critical lesion potentially associated with exposureto AA (EFSA Contam Panel, draft opinion on AA in foods, 2014):

Epidemiological evidence for increased cancer riskassociated with AA exposure?

„ In the epidemiological studies available to date AA intake was not associated with an increased risk of common cancers, including those ofthe gastrointestinal or respiratory tract, breast, prostate and bladder… A

9

the gastrointestinal or respiratory tract, breast, prostate and bladder… A few studies suggested an increased risk for renal cell, endometrial and ovarian cancer ( for the two latter particularly in never-smokers), but theevidence is limited and inconsistent… Occupational studies, withtemporarily higher AA exposures, have not shown consistent increasedrisk for cancer.“

Reaction with proteins (Hemoglobin: Hb-adducts)

� surrogate – biomarker for DNA-adduct formation

epoxide hydrolase

CYP450 2E1

O

NH

Acrylamide: toxification / detoxification

AA ���� GA : mouse > rat > human / mercapturic acid formation: humans > rodents

acrylamide

mercapturic acids

GSH-conjugation

AAMA

DNA damage

DNA-N7-guanine-adduct

(by far predominant )

� apurinic sites

� ring opening

formamidopyrimidine

glycidamide

GAMA

NH2 O

10

10

NH2

O

Oglycidamide

Genotoxicity/ Mutagenicity – a comparison at the level of activated carcinogens

� Glycidamide (GA) the genotoxic AA metabolite,

� preferentially alkylating N7 of guanine

� activated nitrosamine NOZ-2 � N7, O6 of DNA guanine/ pyrimidine/

phosphodiester groups

11[Thielen et al. 2006, Baum et al. 2008]

phosphodiester groups

� Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)

GA a potent mutagen? Comparison of induction of hPRT-mutations in V79 cells

DMSO 1 3 10 100

0

40

80

120

160

control (DMSO) NOZ-2 (1-100 µM)

***

*

[µM]

***

mu

tan

ts /106

ce

lls

DMSO 400 800 1200 20000

40

80

120

160

control (DMSO)

GA (400-2000 µM)

***

***mu

tan

ts /

106

cells

[µM]

***

� NOZ-2: potent mutagen (> 3 µM) � GA: much less potent (>800 µM)

NOZ-2 GA

� NOZ-2: potent mutagen (> 3 µM) � GA: much less potent (>800 µM)

0,1

% D

MSO

3 µM

10 µ

M

30 µ

M

0

20

40

60

80

100

***

mu

tan

ts/1

06 c

ells

BPDE

[Thielen et al. 2006, Baum et al. 2008 ] 12

� BPDE: potent mutagen (> 3 µM)

In vivo studies

(rats)

13

Biomarker response in rats ingesting AA for 9 d in food/water

AA exposure

daily for 9 days in food: 100 µg/kg AA bw/d in food or water

single dose : 1 x 900 µg/kg in water

Food : French fries (sliced:FFS;reconstituted:FFR)

control : drinking water (DW)

Biomarkers

���� Hemoglobin-(Hb-)adducts in blood (AA-Val/GA-Val)

���� Mercapturic acids (MA) in 24 h urine

[Berger et al., 2010] 14

SD rats: 3 animals/group (male,about 200 g)

Acrylamide biomarkers of exposure

(Hb-adducts, mercapturic acids)

AA

tissue

GI-tract AA/GA-Hb-AdductsAA

AA uptake: Drinking water (DW), French Fries (FFS, FFR)

AA + GSH-Add.AA

GA

systemic distribution via blood

urine

liver

kidney

AA + GSH-Add.

AA/GA-Mercapturic acids

GA + GSH-Add.

15

150

200

250

300

350 AA-DW

FFS

FFR

filled shapes: AAVal

open shapes: GAVal

Val ad

du

ct

form

ati

on

[∆∆ ∆∆ p

mo

l/g

Hb

]

300

400

1000

2000

3000

4000 AAVal

GAVal

Val ad

du

ct

form

ati

on

[p

mo

l/g

Hb

]

single high AA-dose (gavage)

Biomarker - Hb-adductsRepeated oral uptake of AA via water / food (9d)

AA for 9 d ����100µg/kg bw p.o., in FFS/FFR

(French fries) or drinking water

1 3 5 7 9

0

50

100

number of days with AA dosing

Val ad

du

ct

form

ati

on

[

DW 0,45 mg/kg bw 0,9 mg/kg bw 10 mgAA/kg bw0

100

200

Val ad

du

ct

form

ati

on

[p

mo

l/g

Hb

]• Hb-Adducts : Monitored 24h after respective last AA intake

���� AA-Val increases with cumulative AA uptake (linear with time);

���� GA-Val : no difference to untreated controls at any dosage

(9 x100µg/kg / 1x 900 µg/kg)

[Berger et al., Mol Nutr. Food Res 2010] 16

Biomarker– mercapturic acids in urine

Oral uptake via water / food

AA/GA-mercapturic acids, in 24h urine

1,2 1 day

3 days

� ~50% of applied dose excreted as

mercapturic acids (AAMA+GAMA)

150

175

200

225

250

am

ou

nt

excre

ted

[n

mo

l]

Water - AAMA

Water - GAMA

Food - AAMA

Food - GAMA

Control - AAMA

Control - GAMA

150

175

200

225

250

am

ou

nt

excre

ted [

nm

ol]

�Bioavailability of AA in foods comparable to drinking water (d5:accidental overdose)

AA-DW FFS0,0

0,2

0,4

0,6

0,8

1,0

GA

MA

/ A

AM

A

3 days 5 days

7 days

9 days

ratio:

GAMA / AAMA

[Berger et al., Mol Nutri Food Res 2010] 17

1 3 5 7 90

25

50

75

100

125

am

ou

nt

excre

ted

[n

mo

l]

number of days with AA dosing

1 3 5 7 90

25

50

75

100

125

am

ou

nt

excre

ted [

nm

ol]

number of days with AA dosing

• no marked differences in bioavailability between water and food

• urinary mercapturic acids (AAMA and GAMA): clear indication for

metabolic GA formation in the liver

• no significant increase in GA-Hb adducts up to total dose of 900µg/kg bw(repeated or single dosage)

Biomarker response in rats: repeated oral

uptake of AA via water / food

(repeated or single dosage)

Conclusion

[Berger et al., Mol Nutr Food Res 2010] 18

At AA-dosage of 100µg/kg b.w./ day:

any GA formed from AA is effectively coupled to GSH in rat liver

Formation of phase I / II metabolites in primary rat hepatocytes: GA versus AA-GSH

19[Watzek et al., Arch Toxicol, 2013]

� AA-GSH formation at all AA concentrations faster than GA formation (1.5-3x in medium)

� at 2000 µM AA: steep GA increase at 8h to 16h ���� GSH depletion?

� only at this AA concentration ���� DNA N7-GA-Guanine detected (Cmax=16 h)

AA: single oral dose-response study in rats 0.1 – 10 000 µg/kg bw

Design

� Female SD rats (n = 54; age 50 days, weight about 150-170g), kept on AA-minimized experimental diet ( AA <0,5 µg/kg ���� uptake ≤ 0,08 µg/kg bw/d) for two weeks prior to start and during experiments with free access to (exp.) diet and water

� Low dose range ( 0 – 100 µg/kg bw, gavage):� 8 rats per group : 0, 0.1, 1, 10, 100 µg 1-14C-AA/kg bw

� High dose range (500 – 10,000 µg/kg bw, gavage): � 3 rats per group: 500, 1000, 3000, 6000, 10000 µg AA/kg bw

� Sacrifice 16 h after administration; urine collected; liver, lung, kidney samples taken;

� ���� biomarkers: mercapturic acids in urine; N7-GA-Gua DNA adducts in tissues

20[Watzek et al., Chem. Res. Toxicol., 2012]

Mercapturic acids and N7-Ga-Gua adducts

10

100

1000

10000

100000

sum

of

me

rcap

turi

c a

cid

s [

nm

ol]

AAMA

GAMA

50

60

70

80

90

100

150

200

250

300

7-G

A-G

ua a

dducts

/ 1

08 n

ucle

otides liver

kidney

lung

Mercapturic acids (MA) DNA- N7-Ga-Gua adducts

*

*

*

*

**

**

**

**

**

**

[Watzek et al., Chem. Res. Toxicol., 2012] 21

Kontrolle 0,1 1 10 100 500 1000 3000 6000 100000,1

1

10

sum

of

me

rcap

turi

c a

cid

s [

nm

ol]

AA dose [µg/kg bw]

cont

rol

0,1 1 10 100

500

1000

3000

6000

1000

0

0

10

20

30

40

50

u. Ng.u. Ng.

N7-G

A-G

ua a

dducts

/ 1

0

AA dose [µg/kg bw]

**

*

**

*

MA (AAMA/GAMA):control: ���� background signal

0.1 µg/kg b.w.: no difference to control≥ 1 µg/kg b.w.: clear dose dependence

DNA-N7-GA-Gua: LOD = 0,15 add/108 ncts; LOQ = 0.25; 0.1 µg/kg bw : < LOD ; 1-10 µg/kg bw : <2 add/108 ncts

Up to100 µg/kg bw : no dose related response

N7-GA-Gua adducts in tissues of rats 16 h after AA dosage (via gavage)

1,25

1,50

1,75

2,00

2,25

2,50

7-G

A-G

ua a

ddu

cts

[add

ucts

/10

8 n

ucle

otid

es]

liver

kidney

lung

150

200

250

300

7-G

A-G

ua a

ddu

cts

[a

dd

ucts

/10

8 n

ucle

otide

s]

3

6

9

12

15

18

21

24 liver (R = 0,96)

kidney (R = 0,96)

lung (R = 0,97)

cont

rol

0.1 1 10 100

0,00

0,25

0,50

0,75

1,00

1,25

LODLODLOD

N7-G

A-G

ua a

ddu

cts

[add

ucts

/10

AA-dose [µg/kg bw]

LOD: 0.15 N7-GA-Gua /108 nclt (8 fmol / µmol Gua);LOQ: 0.25 N7-GA-Gua /108 nclt (13 fmol / µmol Gua)

Mean+/-SD;n=8

Dose range: 0 - 100 µg/kg bw Dose range: 0 - 10000 µg/kg bw (kidney)

0,1 1 10 100 1000 10000

0

50

100

150

N7

-GA

-Gu

a a

ddu

cts

[a

dd

ucts

/10

AA-dose [µg/kg bw]

0 200 400 600 800 1000

0

[Watzek et al., 2012] 22

„Background“ human DNA damage

Human „background“ DNA damage:

adducts/108

nucleotidesfmol/µmol guanine

tissue [human]

8-Oxo-dGuo Epe, 2002 100 5330 lymphocytes

7-(2´-Carboxyethyl)guanine Cheng et al., 2010 7 373 liver

N2-ethylidene-dGuo Wang et al., 2006 10 534 liver

7-ethyl-Gua Chen et al., 2007 0,8 42 liver

N2-ethyl-dGuo Wang et al., 2006 0,2 12 liver

1,N2-propano-dGuo Zhang et al., 2006 0,3 15 liver

N2-hydroxymethyl-dA Wang et al., 20107 leucocytes

23

Summary: experimental studies

� GA, the genotoxic metabolite of AA a mutagen of rather modest potency

� Primary rat hepatocytes: rate of AA-GSH formation exceeds rate of GA-formation (F ~ 1.5 -3)

� In vivo study : within dose range 0,1-100 µg AA/kg bw���� no dose related

24

� In vivo study : within dose range 0,1-100 µg AA/kg bw���� no dose relatedincrease of N7-GA-Gua adducts (< 2 adducts/108 nucleotides );levels found close to lower bound background levelreported for similar DNA lesions in human and rat tissues

� Human background DNA lesions ���� point of reference in future riskassessment ?

Acknowledgements

MIT, BostonS.Tannenbaum and his group

Federal Institute for Risk Assessment, BerlinT. Reemtsma, A. Lampen

University of CologneU. Fuhr and his group

TU MunichM. GranvoglIfADo, Technical University of Dortmund

References:Mutagenicity/ GenotoxicityThielen S, Baum M, Hoffmann M, Loeppky RN, Eisenbrand G. Mol Nutr FoodRes. 2006 Apr;50(4-5):430-6.Baum M, Loeppky RN, Thielen S, Eisenbrand G. J Agric Food Chem. 2008 Aug13;56(15):5989-93.

Bioavailability from food:Hemoglobin / Mercapturic acid biomarker responsein ratsBerger F, Feld J, Bertow D, Eisenbrand G, Fricker G, Gerhardt N, Merz KH,Richling E, Baum M. Mol Nutr Food Res. 2011 Mar;55(3):387-99

Dose/Response study: DNA damage and mercapturic acid excretionWatzek N, Böhm N, Feld J, Scherbl D, Berger F, Merz KH, Lampen A, ReemtsmaT, Tannenbaum SR, Skipper P, Baum M, Richling E, Eisenbrand G. Chem ResToxicol. 2012 Feb 20;25(2):381-90

J. G. Hengstler and his groupUniversity of Kaiserslautern

E. Richling F. BergerJ. Feld N. WatzekM. Baum D. Scherbl

25

Financial support by:

Forschungskreis der Ernährungsindustrie (FEI, Germany))

German Federal Ministry of Education and Research (BMBF)

Institute of Scientific Information on Coffee, Switzerland (ISIC)Tchibo GmbH Germany);

Decl.of Interest:Gerhard Eisenbrand serves as scientific consultant toInstitute of Scientific Information on Coffee, Switzerland (ISIC)

And Tchibo GmbH Germany;

Toxicokinetics in rat hepatocytesWatzek N, Scherbl D, Schug M, Hengstler JG, Baum M, Habermeyer M, RichlingE, Eisenbrand G. Arch Toxicol. (2013) 87:1545–1556

Biomarker based human exposure comparison : Acrylamide/ AcroleinWatzek N, Scherbl D, Feld J, Berger F, Doroshyenko O, Fuhr U, Tomalik-ScharteD, Baum M, Eisenbrand G, Richling E. Mol Nutr Food Res. 2012Dec;56(12):1825-37