Differences in the frequency of micronucleated erythrocytes in humans in relation to consumption of fried carbohydrate-rich food

Differences in the frequency of micronucleated erythrocytes in humans in relation to consumption of fried carbohydrate-rich food, Differences in the frequency of micronucleated erythrocytes in humans in relation to consumption of fried carbohydrate-rich food

Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and

Environmental Mutagenesis

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / g e n t o x

C o m m u n i t y a d d r e s s : w w w e l s e v i e r c o m / l o c a t e / m u t r e s

Differences in the frequency of micronucleated erythrocytes in humans in

relation to consumption of fried carbohydrate-rich food

Lilianne Abramsson-Zetterberg a , ∗ , Anna C Vikstr ¨om b ,

Margareta T ¨ornqvist b , Karl-Erik Hellen ¨as a

aLivsmedelsverket, National Food Administration, Toxicology Division, Box 622, 751 26 Uppsala, Sweden

bDepartment of Environmental Chemistry, Stockholm University, 106 91 Stockholm, Sweden

a r t i c l e i n f o

Article history:

Received 3 December 2007

Received in revised form 8 February 2008

Accepted 12 March 2008

Available online 7 April 2008

Keywords:

Micronucleus

Transferrin-positive reticulocytes

Hemoglobin-adducts

Human

Acrylamide

Carbohydrate-rich food

a b s t r a c t

The aim of this study was to investigate if consumption of ordinary carbohydrate-rich food prepared in different ways has an impact on chromosome stability, i.e., on the formation of micronucleated young erythrocytes in humans

Twenty-four persons, divided into two groups, participated during 4 days in a semi-controlled food-consumption study One group (low-heated-food-group, LowHF-group) consumed only food boiled in water (max 100◦C) and the other group (high-heated-food-group, HighHF-group) consumed preferen-tially strongly heated (fried) food From each of the subjects, blood samples were drawn, before and after

4 days The frequency (f) of micronucleated (MN) very young erythrocytes (transferrin-positive

reticu-locytes, Trf-Ret), fMNTrf-Ret, was determined, and the difference in the frequency, before and after the eating period, was calculated The obtained mean differences for the two groups were compared As an indicator of highly heated food the acrylamide (AA) content in part of the consumed foodstuffs was anal-ysed by use of LC/MS–MS and the AA intake estimated In the blood samples the hemoglobin-adduct levels from AA were analysed as a measure of the internal AA dose

The differences between the mean fMNTrf-Ret, before and after the eating period, were−0.15‰ for the

LowHF-group and +0.17‰ for the HighHF-group, p < 0.005 (t-test, one-tailed) The mean total AA intake in

the HighHF-group during 4 days was estimated to about 3000± 450 ␮g per person For the LowHF-group, the mean AA intake was low, 20± 10 ␮g per person

The lowest dose of AA that caused a significant increase of micronucleated erythrocytes in mice is more than a hundred times higher than the AA level in this study Thus, it is unlikely that the exposure to AA is the major cause behind the observed difference The answer is probably to be found in other compounds produced at the same time during heating of the food

© 2008 Elsevier B.V All rights reserved

1 Introduction

Probably over an open fire originally, humans have since ancient

times used heat to prepare foods They likely sought to make

food more tasty, durable, and more easy to masticate Not until

the time of Louis Pasteur did it become clear that the improved

durability obtained by heating foods was due to the killing of

micro-organisms Today’s cookbooks are filled with recipes for preparing

all sorts of dishes using heat Not the least popular methods for

heating food are barbecuing and deep-frying, with French fries and

potato chips being examples of common products that are highly

heated.

∗ Corresponding author Tel.: +46 18 17 57 63; fax: +46 18 17 14 33

E-mail address:liab@slv.se(L Abramsson-Zetterberg)

Unfortunately, the heating of food has also an adverse effect: polycyclic aromatic hydrocarbons (PAHs), heterocyclic aromatic amines (HAAs), 3-monochloropropanediol (3-MCPD), furan, ethyl carbamate, and acrylamide (AA) are examples of unwanted com-pounds for which the concentrations increase with increasing cooking temperature PAHs are formed and released during incom-plete pyrolysis or combustion of organic material A large part of the PAH contamination of food comes with the flames from the seat of the fire High concentrations of individual PAHs have been

muta-genic compounds is the group of HAAs, among which more than twenty different compounds are identified today Most of the HAAs

carba-mate, and 3-MPCD have a somewhat different origin than HAA and PAH Furan can be formed in many foods from such common food constituents as ascorbic acid or polyunsaturated fatty acids, at the 1383-5718/$ – see front matter © 2008 Elsevier B.V All rights reserved

Blood sam

Blood sam

Crisp br

Crisp br

Crisp br

Blood sam

Blood sam

◦C)

volatility of furan, heat-processed canned and jarred foods

of ethyl carbamate and 3-MCPD may in both cases include pre-cursors formed by yeast fermentation (e.g., ethanol and glycerol, respectively), and these compounds are consequently present in

been found in carbohydrate-rich food such as potatoes and cereal products that contain both free asparagine and reducing sugar.

The occurrence of AA in food was first published by Tornqvist

for laboratory animals Two years later they published data about

as 1994 the genotoxicity of AA was verified by IARC (International Agency for Research on Cancer, WHO organ) and the compound

AA is probably not the only genotoxic chemical that is pro-duced when heating carbohydrate-rich foodstuffs There may be other chemicals that have not yet been studied and that may be of equal concern The EU-funded Heatox project (Heat-induced food toxicants, identification, characterisation and risk minimisation at http://www.heatox.org ) has recently listed around 800 compounds

A positive correlation in cancer epidemiology is seen as an ulti-mate proof of carcinogenicity for a given factor in humans Several epidemiological studies published during the period 2003–2007 show no positive association between dietary intake of AA or selected AA-containing foods and increased risks for cancer [17–21] However, a recently published study did find a positive association between dietary AA intake and an increased risk for cer-tain types of cancer (uterus and ovary) in postmenopausal women

dietary AA may increase the cancer risk in humans, it does not establish an unequivocal causal relationship as such However, the sensitivity of epidemiological studies is sometimes too low to detect the relatively moderate cancer risk increment from AA (as

In epidemiological studies the AA intake is often estimated from food-frequency questionnaires The reported intake of AA can be verified by measuring hemoglobin-adducts of AA (Hb-AA), which

is a biomarker for the internal dose over the last months Several studies have been published that show a weak correlation between

Biomarkers of effects, e.g., micronucleated cells in humans are

a helpful tool when the sensitivity of epidemiological studies is

the very youngest erythrocytes (transferrin-positive reticulocytes (Trf-Ret), in peripheral blood), it is possible to detect micronu-cleated reticulocytes in humans The occurrence of transferrin receptors on the surface of the very youngest erythrocytes and the use of antibodies make this separation possible Furthermore, a flow cytometer instead of a microscope allows analysis of many cells in the same experiment, which is a prerequisite for a high sensitivity Half a million cells is a common number of analysed Trf-Ret from each sample.

In the present study we have analysed and compared the frequency of micronucleated Trf-Ret (fMNTrf-Ret) between two groups of persons in a semi-controlled food-consumption study,

Table 2

This table shows the estimated total intake of acrylamide (AA) during the eating period of 4 days, the level of hemoglobin AA-adducts (Hb-AA, in pmol/g globin), the number

of transferrin-positive reticulocytes (Trf-Ret) analysed, the number of micronuclei (MN) registered, and the MN frequency (fMN) (‰) both before and after the eating period

Low-heated-food

High-heated-food

Also listed are the differences in Hb-AA and fMN before and after Id = identification of study participants.**p < 0.005 and***p < 0.0005 (Student’s t-test, one-tailed) When the

smokers (indicated by◦) are excluded from the calculation, the mean fMN difference between the two groups is still significant, p < 0.01 (Student’s t-test, one-tailed).

one group eating medium-heated (max 100◦C) food and one

eat-ing preferentially highly heated food In addition, Hb-AA levels were

measured to verify the difference in AA intake between the groups.

2 Material and methods

2.1 Chemicals

2.1.1 Analysis of acrylamide in food

Acrylamide (CAS 79-06-1) (assay (GC)≥ 99.9%) was obtained from Merck,

Darm-stadt, Germany; deuterium-labelled acrylamide-d3 was obtained from Polymer

Source Inc Dorval, Quebec, Canada; the columns used for solid-phase extraction

(SPE-columns) were Isolute Multimode, and ENV+, from IST, Hengoed, Mid

Glam-organ, UK, the analytical LC column was Hypercarb (5␮m, 50 mm × 2.1 mm) from

Thermo Electron Corp., UK

2.1.2 Analysis of PAH in food

Internal standard was per-deuterated PAH-II from Nist, Standard Reference

Material 2270 containing Pyrene-d10, Perylene-d12, Benzo(ghi)perylene-d12 (LGC

Promochem), Chrysene-d12 from Dr Ehrenstorfer-Sch ¨afers (LGC Promochem) PAH

standards were PAH–mix 68 from Dr Ehrenstorfer-Sch ¨afers (LGC Promochem)

Benzo(j)fluoranthene, 5-Methylchrysene, Dibenzo(a,l)pyrene, Dibenzo(a,i)pyrene,

Dibenzo(a,l)pyrene, Dibenzo(a,h)pyrene, Benzo(c) fluorene, from Dr

Ehrenstorfer-Sch ¨afers (LGC Promochem) Injection standard: bb-binaphthyl (external standard)

in cyclohexane (0.04␮g/ml)

2.1.3 Micronucleus test

Magnetic beads (CELLectionTMPan Mouse IgG Kit) were purchased from Dynal,

Oslo, Norway The mouse anti-human CD71 antibody was purchased from

DakoCy-tomation, Glostrup, Denmark The fluorescent dye Hoechst 33342 was purchased

from Sigma, Aldrich, Sweden, and Thiazole Orange from Molecular probes, Eugene,

Oregon, USA

2.1.4 Analysis of hemoglobin-adducts from AA

Pentafluorophenyl isothiocyanate (>95%, Fluka, Buchs, Switzerland) was

puri-fied on a Sep-Pak silica cartridge[31] Myoglobin, from horse skeletal muscle

(Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany) was precipitated in acidic

acetone solution Formamide (Scharlau Chemie S.A., Barcelona, Spain) was purified

by extraction with n-pentane All other chemicals used were of analytical grade.

2.2 Design of the study

Twenty-four healthy persons, 24–60 (mean = 46) years old, participated in this study Five were smokers The subjects were randomly divided into two groups, 7 women and 5 men in each of the two groups Each of the subjects donated blood twice, before and after an “eating period” of 4 days One of the groups (low-heated-food-group, LowHF-group) ate during 4 days medium-heated (max 100◦C) and fresh vegetables and the other group (high-heated-food-group, HighHF-group) eating preferentially strongly heated (fried) and fresh vegetables The menus are shown

the blood samples

As an indicator of heated food the AA levels in part of the foodstuffs consumed were analysed The subjects belonging to the LowHF-group were instructed to min-imize the consumption of high-heated food during the study and the other group, the HighHF-group, should choose preferentially fried food Since all participants worked at the same place, it was possible to eat the lunches together and see that the instructions were followed Concerning the food eaten at home, before and after work, the subjects belonging to the HighHF-group were supplied with a certain amount of frozen French fries, potato crisps, biscuits, and crisp bread The other group, the LowHF-group, was supplied with fresh potatoes, buns, and white bread One package of each of the supplied foodstuff (for both groups) was kept at the working place for analysis of the AA content All participants kept a food diary over

4 days The reported consumption of vegetables and fruits were about the same in both groups Concerning coffee, almost all participants reported daily consumption Since the participants in the LowHF-group were instructed to keep the coffee intake low, the consumption of coffee in this group was less than in the HighHF-group

To estimate the AA intake during these 4 days, all lunches were pooled each day (i.e., for each day 2 meals, one aimed for the LowHF-group and one for the HighHF-group) and the homogenated pools were separately analysed by LC/MS–MS at the National Food Administration (Table 3) The AA content in all other foodstuffs sup-plied to the two groups, to be eaten at the working place or at home, were analysed separately Some of the foods (two dishes of potato crisps, two dishes of French fries, one dish of boiled and one of fried sausage) were also analysed for PAH

The study has been approved by the Ethics Committee, Faculty of Medicine, Uppsala University (Dnr Ups 02-094)

2.3 The micronucleus assay in human transferrin-positive reticulocytes, Trf-Ret

Part of the blood samples was prepared for analysis of micronucleated

erythro-test, the cells were prepared in accordance with earlier described methods[30,32].

In brief, from each participant a mean of 1.5 ml whole blood was washed and mixed

with 45␮l of a suspension of magnetic beads (CELLection TM Pan Mouse IgG Kit)

pre-coated with mouse anti-human CD71 antibody Since it is only the very youngest

erythrocytes and part of the nucleated cells that express CD71 antigen on the cell

sur-face, these cells are captured with the antibody After the enrichment procedure of

the Trf-Ret, these cells were removed from the beads by DNAse The collected cells

(Trf-Ret) were sphered with 400␮l detergent solution (SDS) during one min and

fixed with about 3 ml freshly prepared 2% formalin in PBS at room temperature The

cells were stored in the fixation solution at room temperature for 4 months Finally,

the day before the flow-cytometer analysis, all cell samples were centrifuged and

the pellet was re-suspended in 0.5 ml fresh staining solution (60 ml PBS with 14␮l

Hoechst 33342, HO342, (500 mM) and 12␮l Thiazole Orange, TO, (1 mg/ml) The

tubes were incubated at 37◦C for 60 min The stained samples were left overnight

at +4◦C

The coded and stained samples were analysed at a rate of 500–1000 cells/s on

a FACSVantage SE flow cytometer (BD Immunocytometry systems, Sunnyvale, CA)

equipped with an argon-ion laser (Enterprise II, Coherent, Santa Clara, CA) operating

at both multiline 350 (UV) and 488 nm For each analysed cell in the flow cytometer,

information about the size, structure, and amount of emitted light (from the

DNA-and RNA-binding dyes) is transformed to electric signals Signals for forward scatter

(FSC, linear), side scatter (SSC, log), TO fluorescence (log), and HO342 fluorescence

(log) were collected A mean of 550,000 Trf-Ret were analysed per sample CellQuest

software (BD) was used for data acquisition

2.4 Analysis of hemoglobin-adducts

The analysis of Hb adducts from AA (Hb-AA) was performed according to the

N-alkyl-Edman method[33] Briefly, globin was first precipitated from

erythro-cytes Then samples of 50 mg globin were dissolved in formamide (1.5 ml) and

derivatised with pentafluorophenyl isothiocyanate (8␮l) to detach the N-terminal

valines, to which AA binds The detached derivative, the

pentafluorophenylthio-hydantoin (PFPTH) of N-(2-carbamoylethyl)-valine was isolated and purified by

extraction The quantification of the level of the Hb adducts from AA was

per-formed by gas chromatography/tandem mass spectrometry (GC–MS/MS) in the

negative ion/chemical ionisation mode, using corresponding deuterium-substituted

PFPTH analyte, synthesised from (2H7)valine, as internal standard Reference globin

with known adduct level from AA to N-terminal valine was used for

calibra-tion [34] The calibration curve (R2= 0.992) included 6 samples in the range

of 0–400 pmol/g globin The limit of quantification (LOQ) was estimated to be

5 pmol/g globin

2.5 Analysis of the acrylamide content in the food

Analysis of AA was performed by liquid chromatography tandem

mass-spectrometry (LC–MS/MS) using electrospray ionisation Briefly, homogenised

samples were extracted with water at room temperature, and deuterium-labelled

AA was added as an internal standard Two types of solid-phase extraction columns

were used to achieve an extract pure and concentrated enough to enable reliable

quantification of AA in solid matrices down to 5␮g/kg[35] The method was

sub-jected to a multi-laboratory collaborative trial validation study including bakery

ware and potato products with AA contents in the range 20–9000␮g/kg[36] It was

concluded that the method was unbiased, and the method precision (CV of 3–9% and

5–13% for repeatability and reproducibility, respectively) was better than prescribed

by international standards

2.6 Analysis of the PAH content in the food

Different PAHs were separated by gas chromatography using a capillary

col-umn and quantified through mass spectrometric detection (GC–MS) Samples were

saponified with potassium hydroxide, extracted with cyclohexane After

wash-ing the extract with methanol/water it was purified on two SPE silica columns

prior to GC–MS analysis The method is based on published procedures[1,37]

with some modifications The applied method was validated for meat, fish, and

vegetable oil in the measurement range 0.3–40␮g/kg The validation included

the 16 different PAHs listed by the EU Commission[38]and some other

com-pounds

2.7 Statistical methods

The differences in fMNTrf-Ret (fMN-diff) before and after the eating period were

calculated for each of the participants The obtained mean fMN-diff for the two

groups (HighHF- and LowHF-group) were then compared with a Student’s t-test.

A comparison was also made (fMN-diff) with the Student’s t-test between the two

groups with no smokers included The same Student’s t-test procedure was used

Fig 1 The difference, in each participant, between the frequencies of

micronucle-ated transferrin-positive reticulocytes (fMN) before and after an eating period of

4 days The participants were divided over two groups, LowHF- (low-heated-food-group) and HighHF-group (high-heated-food-(low-heated-food-group) The LowHF-group ate during 4 days only boiled (max 100◦C) carbohydrate-rich food and the other group (HighHF-group) ate preferentially strongly heated (fried) carbohydrate-rich food The fMN

differences in the two groups are significantly different from each other, p < 0.005 (Student’s t-test, one-tailed) When the five smokers (◦= smokers) were excluded from the calculation the mean fMN differences were−0.13‰ in the LowHF-group

and +0.10‰ in the HighHF-group which also is significant, p < 0.01 (Student’s t-test,

one-tailed)

3 Results

The mean differences between the frequencies of micronucle-ated Trf-Ret, fMNTrf-Ret, for each person, before and after the

are significantly different from each other, p < 0.005 (t-test,

one-tailed) When the five smokers were excluded from the calculation

significant, p < 0.01 (t-test, one-tailed) The mean fMNTrf-Ret for

both groups together (LowHF and HighHF) were 1.42‰ before and 1.43‰ after the eating period These frequencies are in the normal

and the fMNTrf-Ret before the eating period started were 1.29‰ and

Excluding the smokers from the calculation, the fMNTrf-Ret were

French fries included in the lunch meals eaten at the working place

the French fries consumed at home was calculated The AA levels in

AA levels in all foods supplied to the LowHF-group were below

All AA results obtained in the present study were within the

The total AA intake for each of the participant in the HighHF-group

which the intake from potato crisps and French fries contributed

eating period, to which coffee contributed about 50% For both of the groups the consumption of high- or medium-heated food was evenly distributed between 4 days.

The energy intake among the participants in the HighHF-group

compared to the LowHF-group was higher, about 2700 kcal/day vs.

1900 kcal/day, respectively This was mostly due to the different fat

intake The estimated mean fat intake was about 110 g/day for the

HighHF-group and 40 g/day for the LowHF-group The estimated

daily protein intake was about the same for the two groups The

mean carbohydrate intake was slightly higher in the HighHF-group

than in the LowHF-group, about 330 g/day vs 280 g/day,

respec-tively.

The difference in AA intake between the two groups was

veri-fied by measuring Hb-AA adducts The Hb-AA levels were measured

( Table 2 ) Excluding the data from smokers the corresponding

In six food samples, two samples of French fries, two samples

of potato crisps, one sample of fried potatoes with sausage, and

one sample with boiled potatoes and boiled sausage, the PAH

con-tent (the sixteen EU priority PAHs) were measured None of the

quantification level.

4 Discussion

In the present study the difference in the micronucleus

fre-quencies in transferrin-positive reticulocytes (fMNTrf-Ret) before

and after the eating period, was measured for each person There

was a clear and significant difference between the two groups,

the HighHF- and LowHF-group What is the explanation to this?

Comparing our result with earlier data collected from animal

stud-ies with exposure to pure AA, it is not likely that the difference

in intake of AA is the only factor that has caused the difference

in the frequency of micronucleated cells between the two groups

(HighHF- and LowHF-group) From published data, using the

sen-sitive flow cytometer-based micronucleus assay, the lowest dose

of AA that caused a significant increase of micronucleated

ery-throcytes in mice is more than a hundred times higher than the

that the metabolism of AA to the genotoxic agent glycidamide is

expo-sure to AA as the major cause behind the observed difference

seems even more unlikely The answer is probably to be found in

Table 3

Acrylamide (AA) levels in all foods supplied to the study participants

AA concentration in different foodstuffs/dishes

Analyses were made using an LC–MS/MS method with a quantitation limit (LOQ) at

5␮g/kg The different foodstuffs included in the lunch meals were combined prior to

homogenisation and analysis, with the exception of French fries and boiled potatoes

which were analysed separately Dinner meals prepared at home were not analysed

the cascade of compounds that are produced at the same time as AA.

To our knowledge no studies have been published about the rela-tionship between consumption of highly heated carbohydrate-rich food and micronucleated cells or cells with chromosome aber-rations But as early as 1980 Springarn et al [44] measured a

mutagenic effect in vitro of compounds produced in fried

pota-toes, biscuits, and roasted bread The surfaces of the products were removed after heating and extracted with acidic water The extracts from all of the products disclosed a mutagenic effect in the Ames test Without adding S9-mix to the system no effect was found The authors suggested that the compounds responsible for the effect were heteroaromatic reaction products formed in heat-induced reactions of sugars and amino acids Three years later, 1983, Osman

study was that a mutagenic effect remained although the extraction procedure was slightly altered.

AA has been tested several times for mutagenicity The results from these studies point to no or weak activity in the Ames test [46,47,13] On the other hand the clastogenic properties are

the extract could be an important contributor to the positive effect

in the Ames test It is more likely that also other genotoxic com-pounds contributed to the result The answer may be found among all the Maillard reaction products Furthermore, molecular mod-elling has been done to identify and prioritise the heat-generated

Since HAA, PAH, 3-MCPD and furan all are examples of well-known compounds that are produced when food is heated, one may suspect that they have an impact on the result obtained in this study However, the PAH contents of the analysed samples were low Thus it is not likely that PAH contribute to the results obtained Neither are the possible amounts of HAA an explanation, since the precursors for the formation of many HAA originate from meat, and also because the optimum temperature for formation of

3-MCPD and furan are both carcinogenic in animal studies although the underlying mechanism is not clear In a report published by EFSA (European Food Safety Authority) it is concluded that the carcinogenicity of furan is probably attributed to a genotoxic

hand, a later published micronucleus study in vivo did not show

however, which may be the mother substances of free 3-MCPD,

impact of these compounds and its effect on the result obtained in this study is unknown.

It is difficult to relate a biological effect to exposure of a single agent in a mixture of compounds like heated food This concerns both experimental studies and epidemiological studies Further-more, when toxic compounds are produced in many different types

of foodstuff, it may be difficult to find sufficiently large differences

in exposure between study groups, which is a prerequisite in epi-demiological studies This makes it important to collect information from short-term studies with sensitive methods for the analysis of genotoxic endpoints, and using chemical biomarkers for the mea-surements of internal dose In this study, a blood sample was taken from each individual before and after the eating period, which is

a study design that increases the sensitivity of the test Also, using the flow cytometer-based micronucleus assay, the number of anal-ysed cells is high, here almost 600,000 young erythrocytes (Trf-Ret) from each individual at each sampling occasion The high sensitivity

of the flow cytometer-based assay has earlier been demonstrated

in several studies [32,56,57] With sensitive methods it is possible

to detect relatively small effects, effects with an almost negligible

impact at the individual level The change in fMNTrf-Ret found in

this study was within the background variation, normally found in

The Hb-adduct levels have been used as a measure of internal

dose of electrophiles This approach has been used in studies of AA

study, adducts from AA to the N-terminal valine in Hb were used

to measure exposure and internal dose from the AA intake via food

during the test period The change in adduct levels during the test

period was then a confirmation of the different AA exposures in the

the smokers in the study have higher AA-adduct levels This is in

of cigarettes smoked per day and the Hb-adduct levels of AA.

There are weaknesses in all studies, also here The possible

impact from a variety of nutrient contents in the different meals

is not clear Does the higher fat level in the food eaten by the

par-ticipants in the HighHF-group have any impact on the frequency

of micronuclei? Some studies concerning this topic are published.

With the micronucleus assay in lymphocytes the possible

geno-toxic effect of different types of oil was studied Human subjects

were given dietary cis-, trans- and saturated fat during 3 weeks but

vivo studies in mice exposed to different kinds of fat have also been

published In none of these studies was any genotoxic impact found

[62–64] However, in these studies the oil had not previously been

heated to high temperatures On the other hand, although the oil

itself does not have any genotoxic effect it may change the

intesti-nal permeability so that unwanted compounds that may occur in

5 Conclusion

Heated carbohydrate-rich food contains AA and other

com-pounds that together cause an increased level of micronucleated

young erythrocytes in humans Further studies are required to

iden-tify which compounds are most important for this effect.

Acknowledgements

We thank Louise Durling, Ingalill Gadhasson, Birgitta Hellqvist,

Johan Ros ´en, Anders Eriksson, and Elvy Netzel for technical

assis-tance, Qasim Chaudhry, Leif Busk, G ¨osta Zetterberg, Maj Olausson

for useful discussions This work has been carried out with

sup-port from the European Commission, Priority 5 on Food Quality

Research Project), ‘Heat-generated food toxicants – identification,

characterisation and risk minimisation’.

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