Process-induced Toxicants in Food June 3, 2014 Chi-Tang Ho.

Process-induced Toxicants in Food

June 3, 2014

Chi-Tang Ho

From Diet to Disease

High-fat foods are rich in the lipid phosphatidylcholine (PC) and its metabolite choline (C). Intestinal bacteria convert C to TMA. In the liver, the enzyme FMO3 processes TMA to TMAO — a metabolite that makes its way into the blood. Wang et al.1 show that circulating TMAO may contribute to greater plaque development in the arteries, and so to heart disease.

Structure of PC (Phosphatidylcholine)

Active Methyl Donor

Definition of a Process Toxicant

Processing toxicants are defined as those substances present in food as a result of food processing/preparation that are considered to exert adverse physiological (toxicological) effects in humans, i.e., substances that create a potential of real risk to human health.

Trans Fatty Acids

Trans Fatty Acids

Trans fats, unsaturated fatty acids with at least one double bond in the trans configuration

Formed during the partial hydrogenation of vegetable oils

The average consumption of industrially produced trans fatty acids in the US is 2-3% of total calories consumed

Trans Fat regulation

In April 2004, the FDA Food Advisory Committee voted in favor of recommending that trans fatty acid intake level be reduced to "less than 1% of energy (2g per day of a 2000 kcal diet)“

The FDA ruled that, effective January 1, 2006, the nutrition labels for all conventional foods and supplements must indicate the content of trans fatty acids

The New York City has asked 20,000 restaurants and 14,000 food suppliers to eliminate partially hydrogenated oils from kitchens

Typical trans fatty acid contents of foods produced by partially hydrogenated vegetable oils

Food g/serving % of daily energy

intake for 2000-kcal diet

French fries: 4.7-6.1 2.1-2.7 Fish burger 5.6 2.5 Chicken nuggets 5.0 2.3 Pizza 1.1 0.5 Popcorn 1.2 0.5 Doughnuts 2.7 1.2

Intake of trans fat and diseases

Cardiovascular disease: 2% increase in energy intake from trans fatty acids was associated with a 23% increase in the incidence of coronary heart disease Raise levels of low-density lipoprotein cholesterol Reduce levels of high-density lipoprotein cholesterol Increase the ratio of total cholesterol to HDL

cholesterol

Intake of trans fat and diseases

Increase the risk of sudden death from cardiac causes.

May increase the risk of diabetes Trans fats promote inflammation Trans fats may cause endothelial

dysfunction

Produced during oxidation of poly-unsaturated fatty acids

Acrolein, malonaldehyde, and 4-hydroxy-2-nonenal

React with protein and DNA and as a result are toxic and mutagenic.

O

Acrolein Malonaldehyde 4-hydroxy2-nonenal (HNE)

O

OH

Lipid-derived Bioactive Carbonyls Species

CO

CH2

H

O

H

Formation Pathway of Malonaldehyde

L.L

LH

.

O O

.

LH

O O

+ O2+ O2

OOH

O

O

Malonaldehyde

BicycloendoperoxidesHydroperoxy Epidioxides

O O OOH

Pryor et al, 1976

Linolenic acid

←

How is its formation mechanism ?

Acrolein

Strongest electrophile among α,β-unsaturated aldehydes react with thiol and amino groups of protein causing alteration

of the structure and function of matrix protein React with DNA at guanine residues to form 8-hydroxy-

propanodeoxyguanosine (OHPdG)

Generated in biological systems under oxidative stress Environmental and industrial pollutant, automobile

exhaust, wood smoke, cigarette smoke

O

Reaction of acrolein with proteins

Acrolein

Acrelein will produce abundantly through autoxidation of ω-3 polyunsaturated fatty acids, such as fish oil

ω-3 eicosapentaenoic acid (EPA) (20:5)

ω-3 docosahexaenoic acid (DHA) (22:6)

C5H11

CO2H

arachidonic acid

C5H11

CO2H

beta-cleavage of alkoxy radical

HOO

C5H11

O

C5H11

O

OOH

O2

O2

beta-cleavage of alkoxy radical

C5H11O+ O

acrolein

Proposed formation pathway for undesirable acrolein

Reactive Carbonyl Species (RCS) from Maillard Reaction

Deoxyosone, methylglyoxal (MGO) and glyoxal (GO)

Produce through Maillard reaction Strong electrophiles, react with proteins

and DNA

RCS Generation in Vitro

R-NH2 + Glucose

C

H

C

NH

OHH

C HHO

C

C

OHH

OH

CH2OH

H

Schiff's base

HO

HOH2C O

OH OHNH_Protein

H

H

O

O

Glyoxal

C

H

C O

CH2

C

C

OHH

OH

CH2OH

H

O

3-DG

-Glyceraldehyde,

H3C

H

O

O

Methylglyoxal

"Classical" Amadorirearangement

CH

C

HNR

OH

C HHO

C

C

OHH

OH

CH2OH

H

CH2

C

HNR

O

C HHO

C

C

OHH

OH

CH2OH

H

HOO

OHOH

Fructosamine

NHROH

-H2O, -RNH2

-Erythritol orerythrose andH2O2, -RNH2

Maillard Reaction

Methylglyoxal Generation in Vivo

Fatty acid

Acetone

Acetyl-CoA

Fats

Glycerol

Dihydroxyacetone phosphateGlyceraldehyde-3-phosphate

Methylglyoxal

Glycerol-3-phosphateFructose-1,6-bisphosphate

Fructose-6-phosphate

Glucose-6-phosphate

GlucoseProtein

Gly, Thr

Aminoacetone

Aldose reductase

NADPH

D-LactateAdvanced glycation

end products1,2-propanediol

Glyoxalase I

Glyoxalase II

GSH

MG

Protein Glycation

DNA GlycationAGEs

Inflammation

Thrombosis

Angiogenesis

Tissue Injury

Protein

Cross Linking

Cellular

Apoptosis

Gene

Transcription

Ramasamy, R., Yan, S. F., and Schmidt, A. M., 2006, Cell 124, 258-260

Glycation of Transcription Modulators

Changes Caused by Methylglyoxal (MG)

Diabetesretinopathyneuropathy and nephropathy

Non-diabetic nephropathy Macrovascular disease (atherosclerosis) Alzheimer's disease Cataracts Aging

Health Concerns with MG

Human plasma MG level in different studies

MG (μg/dL) Quantifying method† Source

Patients Control

15.84.6 (n=20)4.7 1.2

(n=15)2,3-diaminonaphthalene; 3,4-hexanedione; ESI/LC/MS

Odani, Hinzato, and Matsumoto, 1999

20.63.8 (n*=15)4.9 1.2

(n=15)Methanol; Meso-stilbenediamine; HPLC (358nm)

Khuhawar and Kandhro, 2006

† Quantifying method is listed with the sequence of deproteinization agents, derivatization agents, internal standard, and equipment.

* This study included both diabetes and ketosis patients.

MG in Human Plasma

MG in Beverages

minimum

Mean

Maximum

050

100150200

250300350

400

450

500

MG

ug

/ 1

00

mlMG in Beverages

minimum

Mean

Maximum

One can of soda: 300 mlBlood volume in kid: 2.5 LAvg. MG in Soda: 196 μg/dLMG in one can: 196*3 = 588μgMG Con. in kid: 588/25 =23.5 μg/dLMG Con. in diabetes: 20.6 μg/dL

Consuming soda may increase MG level in Blood

Carbonated Soft Drinks and Carbonyl Stress Burden Thirty minutes after consuming 300 mL of

carbonated cola (11.3 g carbohydrate/100 mL; 7.2 μM MG), the blood MG levels of subjects were raised from 113±22 to 136±34 nM, and the blood glucose levels were raised from 94±8 to 113±18 mg/dL.

Glucose and MG containing carbonated soft drinks appear to lead to transient increase in plasma MG levels. It is of great interest whether habitual intake of carbonated drinks enhances human carbonyl stress. Nakayama et al., J. Toxicol. Sci. 34(6):

699-702, 2009

MG in Commercial Cookies

MG levels in commercial cookies range from 3.7 to 81.4 mg/Kg

Commercial cookies made from ammonium bicarbonate and fructose showed the highest levels of MG

MG was rapidly formed on the upper site of the cookies regardless of shape or thickness of the samples

Dietary exposure of Spanish population to MG from cookies was estimated to be 216 μg/person/day

Arribas-Lorenzo and Morales, 2010Arribas-Lorenzo and Morales, 2010

5.8

17.1

36.7

27.1

45.7

33.8

27.8

63.160.0

66.6

0

10

20

30

40

50

60

70

80

INC GA EC ECG EGC EGCG PY TF1 TF2 TF3

•MG: Polyphenolic compound mixed with molar ratio 3:1 •Incubation of 1 hour

O

OR1

HO

OH

OH

OH

R2

R1 R2

EC H H

ECG Gallate H

EGC H OH

EGCG Gallate OH

Green Tea Catechins

O

OR1

O

OH

HO

HO

OHOR2

OH

OH

OH

O

R1 = R2 = H ; Theaflavin(TF1)

R1 = G , R2 = H or R1 = H , R2 = G ; Theaflavin monogallate esters(TF2)

R1 = R2 = G ; Theaflavin digallate ester(TF3)

G = Galloyl

Black Tea Theaflavins

Inhibition by Tea Polyphenols

Figure 12.

8-Mono-MGOEGCG

O

OH

OH

OH

OH

O

OOH

OH

OH

HO

6

8

O

HO

OHO

O

OH

OH

OH

OH

O

OOH

OH

OH

HO8

OHO

O

OH

OH

OH

OH

O

OOH

OH

OH

HO

6

O

HO

6-Mono-MGOEGCG 6,8-Di-MGOEGCG

MGO× 1 MGO × 2

MGO× 1

O

O

OH

OH

OH

O

OOH

OH

OH

HO

H

OH-

O

O

H

O

O

H

Formation of EGCG-MG Adduct

Another Maillard Reaction-derived Toxicants: Heterocyclic Amines (HAs)

Heterocyclic amines occur at the ppb range in foods

Most of them demonstrated potent mutagenicity and as probably human carcinogens

IQ has even demonstrated carcinogenic activity in monkeys

Their capability of formation even during ordinary cooking practices implies frequent exposure by the general public

Abbreviation Z R1 R2 R3

IQ C H H H

MeIQ C Me H H

MeIQx N H H Me

4,8-DiMeIQx N Me H Me

Commonly occurred Heterocyclic amines

Mechanism for the formation of heterocyclic amines

Mechanism for the formation of 4,8-DiMeIQx

Formation of PhIP: A Powerful Carcinogen in Processed Foods

COOH

NH2

O

x

1

2

12

Compound 1

Compound 2

Phenylalanine

Phenylacetaldehyde

O

O

OH

OH

OH

HO

OHO

OH

OH

OH

O

O

OH

OH

OH

HO

OHO

OH

OH

OH

Phenylethenyl

O

O

OH

OH

OH

HO

OHO

OH

OH

OH

Phenylethenyl

N N

N

CH3

NH2

PhIP

N

NO

CH3

NH2

Creatinine

Postulated Pathways for EGCG’s Inhibitory Activity in PhIP Formation

Factors affect formation of HAs Temperature Time Precursors: creatinine, phenylalanine, (reducing sugars,

amino acids) Involvement of Lipids Direct involvement of Strecker aldehydes Water content Concentration of polyunsaturated fats Metal ions Antioxidants

Acrylamide

Acrylamide - toxicology

Proven neurotoxic compound in animals and in humans

Effects range from drowsiness to incoordination, hallucinations, confusion, abnormal sensation, muscle weakness, incoordination

Genotoxic compound with the potential to affect the germinal cells thus leading to hereditary changes

Causing cancer in laboratory animals (rats) Studies in humans (e.g. 8000 workers in China)

which were positive on neurotoxicity failed to prove relationship with cancer in humans (too small numbers ?)

How do we know ...

... whether somebody had been exposed to acrylamide ?

Acrylamide binds to haemoglobin!

Biomarker: AA-Hb adduct

Level of adduct may reflect exposure to acrylamide over last four months

Research before 1999

“Clear-cut dose-response associations were found between the Hb-adduct levels and peripheral nervous functions symptoms. Thirty-nine percent of those with Hb-adduct levels exceeding 1 nmol/g globin experienced tingling or numbness in their hands or feet. For 23 workers there was strong evidence of PNS impairment due to occupational exposure to acrylamide. All but two had recovered 18 months after the cessation of exposure.”

Sweden: April 2002“ A scientific group at the University of Stockholm ... has found that acrylamide is formed during heating of starch-rich foods to high temperatures.The Swedish National Food Administration has developed a new, rapid method for the analysis of acrylamide in foods.Analysis has shown that acrylamide is present in a large number of foods, including many regarded as staple foods. The levels of acrylamide differ widely within each food group analysed.”

Mechanism for the formation of acrylamide from asparagine through the early Maillard reaction

Chloropropanols

A group of chemical contaminants comprising three carbon alcohols and diols with one or two chlorine atoms that are hypothetically derived from glycerol

Dichloropropanols and chloropropanediols were identified as contaminants of the savory food ingredient acid-hydrolyzed vegetable protein (acid-HVP) in the 1970s and 1980s.

In view of 3-MCPD (3-monochloropropane-1,2-diol) toxicity, the EC has proposed a provisional tolerable daily intake amount of 2 ug/kg body weight/day.

Background– Non-genotoxic carcinogen (JECFA, EU SCF) threshold– Kidney toxicity at chronic exposure– Inhibits male fertility at high doses

Occurrence– Hydrolyzed vegetable proteins (HVP)– Low levels in foods (biscuits, bread, cooked/cured fish and meat)– Migration (food contact materials)

Human dietary exposure– 2 g/person/day from savory foods– 140-1100 g/person/day from soy sauce

EU Restriction of 3-MCPD in process flavor is 20 ppb (liquid base) and 50 ppb (dry base)

3-MCPD (3-monochloropropane-1,2-

diol)

3-MCPD esters

Potential concern — Occurrence of 3-MCPD esters in a wide range of cooked foods

and breast milk (data published 2004 – 2006)

— 3-MCPD-esters in the diet may release some free 3-MCPD by

action of gut lipases, potentially contributing to the overall

dietary exposure to free 3-MCPD

O

R O

O

R O

Cl

O

R O

Cl

OH O

R O

OH

Cl

OH

OH

Cl

R = alkyl

3-MCPD 3-MCPD diesters 3-MCPD monoesters

Proposed mechanism for the formation of 3-MCPD diesters from DAG. L represents lipid.

Published in: Xiaowei Zhang; Boyan Gao; Fang Qin; Haiming Shi; Yuangrong Jiang; Xuebing Xu; Liangli (Lucy) Yu; J. Agric. Food Chem.  2013, 61, 2548-2555.DOI: 10.1021/jf305252qCopyright © 2013 American Chemical Society

Other Process-induced Food Toxicants in Question

Furan

5-hydroxymethyl-2-furfural

Furan in baby foods & infant formula

0

20

40

60

80

100

120

baby foods in glassjarrs (vegetables,

meat, fruit)

baby food (powderfor porridge)

baby beverage(juices & teas)

infant formula

211 4 10 27

co

nc

. (p

pb

)

25%ilemean

medianmax95%ile

75%ile

Potential concernFoods, especially jarred and canned foods, subject to heat treatment can contain furan (in particular baby foods in jars)

- causes liver cancer in animal studies with high potency- genotoxic carcinogen (IARC class 2B ‘possibly carcinogenic to

humans’)- no human epidemiological data on cancer

Furan

ExposureNo reliable exposure Estimates

(~ 1 µg/kg bw/day )

U.S. Food and Drug Administration (May 7, 2004; updated June 7, 2004) (http://www.cfsan.fda.gov/~dms/furandat.html)Reinhard et al., Mitt. Lebensmit. Hyg. 2004, 95, 532-535.

Ascorbic acid is the major furan precursor under thermal conditions

E Erythrose

TAG Threonine+Alanine+Glucose

GA Glycolaldehyde+Alanine

GS Glycolaldehyde+Serine

E TAG GA GS

Maillard type systems

LA Linoleic acid (C18:2)

T Trilinoleate

LnA Linolenic acid (C18:3)

Tn Trilinolenate

LA T LnA Tn

Lipids

AA Ascorbic acid

DAA Dehydroasc. acid

AA DAA0

2x103

4x103

6x103

8x103

1x104

mo

l/mo

l Fu

ran

Ascorbic acid

(Maerk et al., J. Agric. Food Chem. 2006, 54, 2786-2793)