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:
Fish burger
Chicken nuggets
Pizza
Popcorn
Doughnuts
4.7-6.1
5.6
5.0
1.1
1.2
2.7
2.1-2.7
2.5
2.3
0.5
0.5
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

Lipid-derived Bioactive Carbonyls Species



Produced during oxidation of poly-unsaturated
fatty acids
Acrolein, malonaldehyde, and 4-hydroxy-2nonenal
React with protein and DNA and as a result are
toxic and mutagenic.
H
O
CH 2
O
OH
C
O
O
Acrolein
H
Malonaldehyde
4-hydroxy2-nonenal
(HNE)
Formation Pathway of Malonaldehyde
How is its formation
mechanism ?
O
O
+ O2
.
←
LH
+ O2
LH
L
O
O
.
Linolenic acid
.
L
O
OOH
O
OOH
Bicycloendoperoxides
Hydroperoxy Epidioxides
O
O
Malonaldehyde
Pryor et al, 1976
Acrolein

Strongest electrophile among α,β-unsaturated
aldehydes




O
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-hydroxypropanodeoxyguanosine (OHPdG)
Generated in biological systems under oxidative stress
Environmental and industrial pollutant, automobile
exhaust, wood smoke, cigarette smoke
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)
Proposed formation pathway for undesirable acrolein
CO2H
C5H11
arachidonic acid
O2
HOO
CO2H
C5H11
beta-cleavage of alkoxy radical
O
C5H11
O2
O
C5H11
OOH
beta-cleavage of alkoxy radical
O
C5H11
+
O
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
Maillard Reaction
R-NH2 + Glucose
O
H
-Erythritol or
erythrose and
H2O2 , -RNH2
H
H
Schiff's base
HOH2C
HO
H
Glyoxal
C
H C
H
OH
C H
-H2O, -RNH2
O
HO
H
NH_Protein
OH
N
OH
C
OH
H C
OH
CH2OH
R
H
N
OH
NHR
O
HO
OH
OH
HO
H
R
H
N
H
C
C
HO
O
H
C
OH
H C
OH
-Glyceraldehyde,
OH
C H
C
OH
H
C
OH
H C
OH
H C
OH
CH2OH
C
3-DG
C
C H
O
CH2OH
CH2
O
C
CH2
"Classical" Amadori
rearangement
Fructosamine
O
CH2 OH
H3C
O
O
H
Methylglyoxal
Methylglyoxal Generation in Vivo
Protein
Glucose
Fats
Glucose-6-phosphate
Glycerol
Gly, Thr
Fructose-6-phosphate
Aminoacetone
Fatty acid
Acetyl-CoA
Glycerol-3-phosphate
Fructose-1,6-bisphosphate
Acetone
Glyceraldehyde-3-phosphate
Dihydroxyacetone phosphate
Methylglyoxal
Glyoxalase I
Glyoxalase II
Advanced glycation
end products
GSH
D-Lactate
Aldose reductase
NADPH
1,2-propanediol
Changes Caused by Methylglyoxal (MG)
MG
AGEs
Protein
Glycation
DNA Glycation
Inflammation
Protein
Cellular
Gene
Thrombosis
Cross Linking
Apoptosis
Transcription
Glycation of Transcription
Modulators
Angiogenesis
Tissue Injury
Ramasamy, R., Yan, S. F., and Schmidt, A. M., 2006, Cell 124,
258-260
Health Concerns with MG

Diabetes
 retinopathy
 neuropathy
and nephropathy
Non-diabetic nephropathy
 Macrovascular disease (atherosclerosis)
 Alzheimer's disease
 Cataracts
 Aging

MG in Human Plasma
Human plasma MG level in different studies
MG (μg/dL)
Patients
Quantifying method†
Source
Control
15.84.6 (n=20)
2,3-diaminonaphthalene; 3,44.7  1.2 (n=15)
hexanedione; ESI/LC/MS
20.63.8 (n*=15)
4.9  1.2 (n=15)
Odani, Hinzato,
and Matsumoto,
1999
Methanol; Meso-stilbenediamine; Khuhawar and
Kandhro, 2006
HPLC (358nm)
† 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 Beverages
500
450
400
350
300
250
200
150
100
50
0
minimum
Mean
Maximum
Maximum
a
Te
Fr
ui
t
gy
minimum
En
er
at
ed
Mean
ar
bo
n
C
MG ug/
100ml
MG in Beverages
Consuming soda may increase MG level in Blood
One can of soda:
300 ml
Blood volume in kid: 2.5 L
Avg. MG in Soda:
196 μg/dL
MG in one can:
196*3 = 588μg
MG Con. in kid: 588/25 =23.5 μg/dL
MG Con. in diabetes: 20.6 μg/dL
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, 2010
Inhibition by Tea Polyphenols
80
70
66.6
63.1
60.0
Green Tea Catechins
60
50
45.7
40
36.7
33.8
30
OH
27.8
27.1
OH
17.1
20
10
HO
5.8
O
R2
0
INC
GA
EC
ECG
EGC
EGCG
PY
TF1
TF2
TF3
OR1
•MG: Polyphenolic compound mixed with molar ratio 3:1
•Incubation of 1 hour
OH
Black Tea Theaflavins
OH
OR1
OH
HO
R1
R2
R1 = R2 = H ; Theaflavin(TF1)
EC
H
H
R1 = G , R2 = H or R1 = H , R2 = G ;
Theaflavin monogallate esters(TF2)
ECG
Gallate
H
EGC
H
OH
EGCG
Gallate
OH
O
O
HO
O
OH
OH
OR2
OH
R1 = R2 = G ; Theaflavin digallate
ester(TF3)
G = Galloyl
Formation of EGCG-MG Adduct
OH
O
O
H
HO
OH
O
OH
O
H
O
OH
O
O
O
H
OH
OHOH
MGO × 2
MGO× 1
MGO× 1
OH
HO
HO
OH
O
8
OH
O
HO
OH
HO
6
OH
O
OH
HO
OH
8-Mono-MGOEGCG
6
OH
O
O
OH
O
OH
O
OH
O
8
HO
OH
O
Figure 12.
HO
O
O
OH
OH
OH
O
OH
OH
O
OH
OH
OH
6-Mono-MGOEGCG
OH
6,8-Di-MGOEGCG
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
Commonly occurred Heterocyclic amines
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
Mechanism for the formation of heterocyclic amines
Mechanism for the formation of 4,8-DiMeIQx
Formation of PhIP: A Powerful
Carcinogen in Processed Foods
Postulated Pathways for EGCG’s Inhibitory Activity in
PhIP Formation
CH3
CH3
N
N
NH2
COOH
NH2
N
O
Creatinine
N
N
PhIP
NH2
Phenylalanine
x
OH
O
Phenylacetaldehyde
O
HO
2
OH
OH
OH
O
2
OH
Compound 1
OH
O
HO
OH
O
OH
1
OH
Phenylethenyl
1
OH
OH
O
OH
OH
OH
O
O
HO
OH
OH
OH
Phenylethenyl
O
OH
OH
O
OH
Compound 2
OH
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
19
99
“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
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.”
“A
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 (acidHVP) 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.
3-MCPD
(3-monochloropropane-1,2-diol)
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 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
R
OH
R
O
O
Cl
3-MCPD
O
R
O
O
HO
R
O
OH
3-MCPD diesters
R = alkyl
HO
O
Cl
O
Cl
3-MCPD monoesters
Cl
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/jf305252q
Copyright © 2013 American Chemical Society
Other Process-induced Food Toxicants in
Question
Furan
5-hydroxymethyl-2-furfural
Furan
Potential concern
Foods, 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 in baby foods & infant formula
120
Exposure
No reliable exposure
Estimates
(~ 1 µg/kg bw/day )
conc. (ppb)
100
80
25%ile
mean
median
max
95%ile
75%ile
60
40
20
0
baby foods in glass
jarrs (vegetables,
meat, fruit)
baby food (powder
for porridge)
baby beverage
(juices & teas)
infant formula
211
4
10
27
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.
mol/mol Furan
Ascorbic acid is the major furan precursor
under thermal conditions
1x10
4
8x10
3
6x10
3
4x10
3
2x10
3
Maillard type systems
Ascorbic acid
Lipids
0
AA
DAA
AA
Ascorbic acid
DAA Dehydroasc. acid
E
TAG
GA
GS
E TAG GA GS
LA
Erythrose
Threonine+Alanine+Glucose
Glycolaldehyde+Alanine
Glycolaldehyde+Serine
LA
Linoleic acid (C18:2)
T
Trilinoleate
LnA Linolenic acid (C18:3)
Tn
Trilinolenate
(Maerk et al., J. Agric. Food Chem. 2006, 54, 2786-2793)
T
LnA
Tn