GENOTOXICITY AND CARCINOGENICITY

Influence of a fat-rich diet, folic acid supplementationand a human-relevant concentration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine on the inductionof preneoplastic lesions in the rat colon

Petra Nicken • Nicole Brauer • Alfonso Lampen •

Pablo Steinberg

Received: 14 November 2011 / Accepted: 14 February 2012 / Published online: 28 February 2012

� Springer-Verlag 2012

Abstract In the present study, the effect of three con-

troversially discussed risk factors for colorectal cancer, a

fat-rich diet (16% raw fat content), dietary folic acid

supplementation (50 mg folic acid/kg lab chow) and a

human-relevant concentration (0.1 ppm) of the heterocy-

clic aromatic amine 2-amino-1-methyl-6-phenylimidazo

[4,5-b]pyridine (PhIP), either alone or in combination, on

the induction of aberrant crypt foci (ACF) in the colon of

male Fischer 344 rats was analyzed. The mean number of

ACF per rat in the case of the four groups fed a fat-rich diet

tended to be higher than that of the four groups being fed a

standard diet. However, the increase in the mean number of

ACF per rat only reached statistical significance in the case

of the rats receiving a fat-rich lab chow supplemented with

50 mg/kg folic acid. Moreover, a concentration of 0.1 ppm

PhIP per se, either in the standard or in the fat-rich lab

chow, did not lead to an increase in the mean number of

ACF per rat. In conclusion, the present study provides

additional evidence for a colon cancer promoting effect of

folic acid supplementation when rodents are fed the com-

pound in supraphysiological concentrations.

Keywords Aberrant crypt foci � Colorectal cancer �Fat-rich diet � Folic acid � Heterocyclic aromatic amines

Introduction

Colorectal cancer is the third most common cancer in men

(6,63,000 cases, 10% of the total) and the second in women

(5,71,000 cases, 9.4% of the total) worldwide (Ferlay et al.

2010; Jemal et al. 2011). Moreover, about 6,08,000 deaths

from colorectal cancer are estimated worldwide, account-

ing for 8% of all cancer deaths and making it the fourth

most common cause of death from cancer. As early as in

1999, a scientific panel of the World Health Organization

postulated that consumption of red meat and particularly of

processed meat most probably enhances the risk to develop

colorectal cancer (Scheppach et al. 1999). In 2007, a joint

committee of the World Cancer Research Fund and the

American Institute for Cancer Research reviewed the

available scientific literature and concluded that red meat

as well as processed meat is a convincing cause of colo-

rectal cancer (World Cancer Research Fund/American

Institute for Cancer Research 2007). In this regard, it has

been suggested that heterocyclic aromatic amines (HCAs)

being formed when muscle meat is strongly heated may

trigger colorectal cancer development (Sinha et al. 2001,

2005; Shin et al. 2007; Rohrmann et al. 2009; Zheng and

Lee 2009; Cross et al. 2010).

HCAs are pyrolysis products derived from amino acids

or proteins that emerge when protein-rich food is fried,

grilled, or cooked. They are particularly formed on the

surface of strongly heated fish and meat, but low levels of

HCAs have also been detected in white and red wine, in

different beer types and in the tar fraction of cigarettes.

Among the various HCAs known, the quantitatively most

important one in fish and meat samples is 2-amino-1-

methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). PhIP is

mutagenic in bacteria and in mammalian cells (Thompson

et al. 1987) and carcinogenic in the colon of male rats

P. Nicken � N. Brauer � P. Steinberg (&)

Institute for Food Toxicology and Analytical Chemistry,

University of Veterinary Medicine Hannover,

Bischofsholer Damm 15, 30173 Hannover, Germany

e-mail: pablo.steinberg@tiho-hannover.de

A. Lampen

Federal Institute for Risk Assessment, Thielallee 88-92,

14195 Berlin, Germany

123

Arch Toxicol (2012) 86:815–821

DOI 10.1007/s00204-012-0819-1

(Ito et al. 1991; Hasegawa et al. 1993). It should be noted that

in the case of the studies by Ito et al. (1991) and Hasegawa

et al. (1993) as well as in most of the later reports having been

published in the scientific literature and dealing with the

induction of colon carcinomas by PhIP, rats were fed with

extremely high (i.e., totally human-irrelevant) amounts of

PhIP in the range of 100 to 400 ppm. In contrast, in those

feeding studies, in which PhIP was used in human-relevant

concentrations (i.e., \0.1 ppm), no preneoplastic or neo-

plastic lesions were induced (Fukushima et al. 2004; Doi

et al. 2005; Kuhnel et al. 2009), whereby in these three

studies PhIP was added to standard rat chow.

Various studies reported that high levels of dietary fat

were able to enhance colon epithelial cell proliferation,

thereby promoting colon carcinogenesis (Thornton and

MacDonald 1997; Kim et al. 1998; Fujise et al. 2007). In

accordance with these observations Ochiai et al. (1996) and

Ubagai et al. (2002) showed that a fat-rich diet strongly

enhanced the induction of preneoplastic lesions in the

colon of rats fed 400 ppm PhIP (Ochiai et al. 1996; Ubagai

et al. 2002). However, up to now it is not known whether a

human-relevant concentration of PhIP added to a fat-rich

diet could eventually lead to the formation of colonic

lesions.

Folic acid fortification of cereal grain flour was fully

implemented in the United States and Canada in 1998 and

has led to a significant reduction in neural tube defects in

neonates (Berry et al. 2010). In this context, the possibility

that folic acid fortification may enhance the risk to develop

colorectal carcinomas is a matter of considerable debate.

On the one hand, Mason et al. (2007) highlighted a tem-

poral association between folic acid fortification of enri-

ched cereal grains in the United States and Canada and an

increase in the incidence of colorectal carcinomas in these

two countries. Furthermore, in a multicenter study by Cole

et al. (2007), the safety and efficacy of folic acid supple-

mentation (1 mg/day, in part in combination with aspirin)

for preventing colorectal adenomas was analyzed in par-

ticipants with a recent history of colorectal adenomas.

Three to 5 years after beginning folic acid supplementa-

tion, the risk of developing advanced colorectal lesions,

multiple (C3) colorectal adenomas as well as noncolorectal

cancers (prostate cancer) was significantly increased (Cole

et al. 2007). On the other hand, very recent studies show

that folic acid fortification actually reduces the risk to

develop colorectal cancer (Gibson et al. 2011; Kennedy

et al. 2011; Lee et al. 2011; Stevens et al. 2011).

In a limited number of animal studies, the effect of

supraphysiological levels of folic acid on colon carcino-

genesis has been analyzed. Kim et al. (1996) reported a

nonsignificant trend toward increased colorectal tumori-

genesis in Sprague–Dawley rats treated with 1,2-dimeth-

ylhydrazine and fed a supraphysiological dose of folic acid

(40 mg folic acid/kg diet, i.e., 20 times the daily folic

acid requirement of rats). In support of this early finding,

azoxymethane in combination with a dietary folic acid

supplementation exceeding the basal requirements of rats

by 1,000 times (2.0–5.0 g folic acid/kg diet) significantly

increased the number of so-called aberrant crypt foci

(ACF), preneoplastic lesions in the rat colon (Bird 1987;

Tudek et al. 1989), when compared to the combination of

the above-mentioned colon carcinogen and a standard diet

including 2 mg folic acid/kg diet in Fischer 344 rats

(Reddy et al. 1996; Wargovich et al. 1996, 2000). Kim

(2004) hypothesized that in a strongly procarcinogenic

environment, in which the appearance of microscopic

neoplasms is inevitable, exceptionally high folic acid

supplementation may promote the progression of chemi-

cally induced colorectal neoplastic lesions. Up to now, it is

not known whether a supraphysiological level of folic acid

combined with a fat-rich diet and/or a human-relevant

concentration of PhIP will enhance the induction of ACF in

the rat colon.

In the present study, the effect of three controversially

discussed risk factors for colorectal cancer, a fat-rich diet,

dietary folic acid supplementation and a human-relevant

concentration of PhIP, either alone or in combination, on

the induction of ACF in the colon of Fischer 344 rats was

analyzed.

Materials and methods

Chemicals

All chemicals were of reagent grade and from commercial

sources. PhIP was obtained from Albrecht Seidel (Bio-

chemisches Institut fur Umweltcarcinogene, Großhansdorf,

Germany).

Animals and diets

Five to six-week-old male Fischer 344 rats were purchased

from Charles River WIGA (Deutschland) GmbH (Sulzfeld,

Germany) and allowed to acclimatize to the housing con-

ditions for 4 weeks before the start of the experiment. One

animal per cage was held under specific pathogen-free

conditions at a room temperature of 22�C, 40–60% air

humidity, a fixed 16/8 h day and night cycle and free

access to food and water. PhIP was added to a standard

rodent lab chow and a fat-rich rodent lab chow from

Altromin Spezialfutter (Lage, Germany) to give a final

PhIP concentration of 0.1 ppm. The raw fat content of the

standard and the fat-rich lab rodent chow was 5 and 16%,

respectively. The concentration of the various fatty acids in

the standard and fat-rich diet is listed in Table 1. The

816 Arch Toxicol (2012) 86:815–821

123

physiological and supraphysiological concentration of folic

acid in the standard and the fat-rich rodent lab chow was 2

and 50 mg/kg, respectively. All diets were regularly ana-

lyzed for the presence of mutagenic compounds such as

N-nitrosamines and aflatoxins by the manufacturer (Altro-

min Spezialfutter) and were consistently tested negative

throughout the whole experimental period. The animal

study was approved by the local regulatory agency for

animal experiments (Niedersachsisches Landesamt fur

Verbraucherschutz und Lebensmittelsicherheit, Oldenburg,

Germany [approval number 33.9-42502-04-09/1729]).

Experimental design

Rats were randomly divided into 8 groups of 12 animals

each. Group 1 was fed with the standard lab chow (5% raw

fat and 2 mg/kg folic acid), group 2 with the standard lab

chow (5% raw fat, 2 mg/kg folic acid) containing 0.1 ppm

PhIP, group 3 with the lab chow containing 5% raw fat and

50 mg/kg folic acid, group 4 with the lab chow containing

5% raw fat, 50 mg/kg folic acid and 0.1 ppm PhIP, group 5

with the fat-rich lab chow (16% raw fat and 2 mg/kg folic

acid), group 6 with the fat-rich lab chow (16% raw fat,

2 mg/kg folic acid) containing 0.1 ppm PhIP, group 7 with

the lab chow containing 16% raw fat and 50 mg/kg folic

acid, and group 8 with the lab chow containing 16% raw

fat, 50 mg/kg folic acid and 0.1 ppm PhIP. Body weight

and food consumption were recorded weekly. All animals

were killed after 12 months by decapitation following CO2

inhalation. The entire large intestine was removed for

dissection and flushed with cold phosphate buffered saline

solution (PBS) to remove intestinal content. The tissue was

opened longitudinally, washed extensively with PBS, and

fixed in 4% formalin (Roti�-Histofix 4%; Carl Roth,

Karlsruhe, Germany).

Histology

The formalin-fixed tissues were stained with a 0.1% w/v

methylene blue solution in PBS for 10 min. Thereafter, the

tissue was washed with PBS and the number of ACF per

animal was determined by making use of a SZX16 ste-

reomicroscope from Olympus (Hamburg, Germany).

Statistics

The body and liver weight data were subjected to an

analysis of variance, and the statistical significance was

established by using Tukey’s post hoc test. The data on the

number of ACF per animal were analyzed with the Kruskal–

Wallis test and Dunn’s post hoc test.

Results

Six out of 96 rats died before the end of the feeding period

of 12 months (one animal in group 2, three animals in

group 5, and two animals in group 6), the overall mortality

of 6.25% being very low. No statistically significant dif-

ference in food consumption was observed between the

different groups, rats in groups 1–8 consuming 90 ± 8,

89 ± 8, 94 ± 8, 91 ± 11, 86 ± 11, 101 ± 9, 101 ± 10,

and 103 ± 10 g food/week, respectively. Based on the

weekly food consumption, rats fed 0.1 ppm PhIP (i.e.,

those in groups 2, 4, 6, and 8) took up 8.8–10.2 lg PhIP/

rat/week. In the case of the groups 5–8, which had been fed

the fat-rich lab chow, the body weight was 12–21% higher

than in the groups 1–4, which had received the standard lab

chow (Table 2). Furthermore, the mean weight of the rats

in the groups 6–8 was significantly higher than that of the

rats in group 5. The feeding of the fat-rich lab chow led to a

strong fat accumulation in the liver, the mean liver weight

in the groups 5–8 being 2 to 2.4-fold higher than in the

groups 1–4 (Table 2).

Table 1 Raw fat content and concentration of the various fatty acids

in the standard and fat-rich diet

Constituent Content (mg/kg feed)

Standard diet Fat-rich diet

Raw fat 50,843.333 159,967.560

Capric acid C-10:0 2.500 2,302.500

Lauric acid C-12:0 2.500 2,502.500

Myristic acid C-14:0 2.500 11,002.500

Pentadecanoic acid C-15:0 2.500 1,002.500

Palmitic acid C-16:0 2,700.000 31,700.000

Palmitoleic acid C-16:1 2.500 4,502.500

Heptadecanoic acid C-17:0 2.500 1,002.500

Stearic acid C-18:0 1,250.000 10,250.000

Oleic acid C-18:1 10,950.000 37,450.000

Linoleic acid C-18:2 35,050.000 39,800.000

Linolenic acid C-18:3 150.000 150.000

Arachidic acid C-20:0 250.000 2,550.000

Eicosenoic acid C-20:1 250.000 250.000

Eicosadienoic acid C-20:2 250.000 250.000

Arachidonic acid C-20:4 2.500 2.500

Eicosapentaenoic acid C-20:5 2.500 2.500

Behenic acid C-22:0 250.000 250.000

Docosahexaenoic acid C-22:6 2.500 2.500

Tricosanoic acid C-23:0 2.500 2.500

Nervonic acid C-24:1 2.500 2.500

Erucic acid C-22:1 2.500 1,402.500

Butyric acid C-4:0 0.000 3,500.000

Caproic acid C-7:0 0.000 2,000.000

Caprylic acid C-8:0 0.000 500.000

Arch Toxicol (2012) 86:815–821 817

123

The mean number of ACF per rat in the different groups

is shown in Fig. 1. It is evident that the mean number of

ACF per rat in the case of the four groups that received a

fat-rich lab chow tended to be higher than that of the four

groups being fed a standard lab chow. However, the above-

mentioned increase in the mean number of ACF per rat

only reached statistical significance in the case of the rats

receiving a fat-rich lab chow supplemented with 50 mg/kg

folic acid (group 7) (Fig. 1). Moreover, a concentration of

0.1 ppm PhIP per se, either in the standard or in the fat-rich

lab chow, did not lead to an increase in the mean number of

ACF per rat (Fig. 1).

Discussion

Various surveys have shown that the daily uptake of HCAs

in general does not exceed the amount of 1 lg per person

(Rohrmann et al. 2009; Carthew et al. 2010). Because of

this extremely low exposure level, the Senate Commission

on Food Safety of the German Research Foundation (1998)

came to the conclusion that the risk to develop colorectal

cancer due to the presence of HCAs in food is very low.

This early evaluation is supported by a very recent study by

Carthew et al. (2010), in which the calculated ‘‘Margin Of

Exposure’’ (MOE) value for PhIP was extremely high

(i.e., [100.000). The MOE value represents the quotient

between the doses of a compound causing cancer in ani-

mals and the estimated exposure level of humans to that

compound. If the calculated MOE value is lower than

10.000, the European Food Safety Authority (EFSA) con-

siders the carcinogenic risk of a compound to humans to be

high; if it is greater than 10.000, the carcinogenic risk is

supposed to be low. In the present study, feeding a human-

relevant concentration of PhIP either in a standard diet or in

a fat-enriched diet to rats did not lead to an increase in the

number of preneoplastic lesions (ACF) in the rat colon.

Thus, the results obtained herein experimentally support

previous evaluations that human-relevant concentrations of

PhIP alone or in combination with a fat-rich diet is not a

major factor leading to colorectal cancer in humans (Senate

Commission on Food Safety of the German Research

Foundation 1998; Santarelli et al. 2008; Carthew et al.

2010).

The fact that feeding 0.1 ppm PhIP for up to 12 months

did not lead to a statistically significant increase in the

number of ACF per rat is in accordance with previous

reports (Fukushima et al. 2004; Doi et al. 2005; Kuhnel

et al. 2009). In this context, Fukushima et al. (2004) pos-

tulated that a threshold of 50 ppm for the PhIP-mediated

induction of aberrant crypt foci in the rat colon can actually

be defined. Whereas it is evident from this and previous

studies (Fukushima et al. 2004; Doi et al. 2005; Kuhnel

et al. 2009) that a human-relevant concentration of PhIP

alone is not sufficient to initiate colon carcinogenesis, a

Table 2 Body and liver weight of rats fed the different diets for 12 months

Group Raw fat, folic acid and PhIP content of the feed Body weight (g)a Liver weight (g)a

1 5% Raw fat ? 2 mg/kg folic acid 442 ± 26 14.2 ± 0.8

2 5% Raw fat ? 2 mg/kg folic acid ? 0.1 ppm PhIP 429 ± 22 13.7 ± 0.9

3 5% Raw fat ? 50 mg/kg folic acid 444 ± 18 14.6 ± 0.8

4 5% Raw fat ? 50 mg/kg folic acid ? 0.1 ppm PhIP 428 ± 32 14.0 ± 1.5

5 16% Raw fat ? 2 mg/kg folic acid 481 ± 34b 27.2 ± 3.3b

6 16% Raw fat ? 2 mg/kg folic acid ? 0.1 ppm PhIP 519 ± 31b,c 30.0 ± 3.4b

7 16% Raw fat ? 50 mg/kg folic acid 515 ± 27b,c 31.5 ± 2.9b

8 16% Raw fat ? 50 mg/kg folic acid ? 0.1 ppm PhIP 517 ± 26b,c 32.4 ± 3.5b

a Shown is the mean ± standard deviation of 9–12 rats per groupb The mean of the groups 5, 6, 7, and 8 is significantly different from the mean of the groups 1, 2, 3, and 4, respectively (analysis of variance and

Tukey’s post hoc test; p \ 0.001)c The mean of the groups 6, 7, and 8 is significantly different from the mean of the group 5 (analysis of variance and Tukey’s post hoc test;

p \ 0.05)

raw fat (%) 5

0

1

2

3

4

5

5 5 5 16 16 16 16

folic acid (mg/kg) 2 2 50 50 2 2 50 50

PhIP (mg/kg) 0 0.1 0 0.1 0 0.1 0 0.1

***

*

nu

mb

er o

f A

CF

/rat

Fig. 1 Number of aberrant crypt foci (ACF) per rat after a 12-month

feeding period. The results are expressed as means ± SD of 9–12 rats

per group (statistically significant difference between the means of the

two groups, Kruskal–Wallis test and Dunn’s post hoc test; *p \ 0.05;

**p \ 0.01)

818 Arch Toxicol (2012) 86:815–821

123

mixture of food contaminants (e.g., HCAs, polycyclic

aromatic hydrocarbons, nitrosamines, acrylamide) could

very well overcome the threshold to induce preneoplastic

lesions in the colon. Further experiments are needed to

clarify this important open question in chemically induced

colon carcinogenesis.

The fact that the number of ACF pro rat in all four

experimental groups having been fed a fat-rich diet was,

although not statistically significant, higher than the num-

ber of ACF pro animal in the four experimental groups

receiving a standard lab chow suggests that a fat-rich diet

per se may support the development of ACF. In this con-

text, it has indeed been demonstrated that a fat-rich diet

strongly enhances the number of ACF in rats fed extremely

high concentrations of PhIP (400 ppm; Ochiai et al. 1996;

Ubagai et al. 2002) or azoxymethane (Nigro et al. 1975;

Bull et al. 1979). However, as shown in the present study, a

fat-rich diet does not lead to an increase in the number of

ACF pro animal when these are concomitantly fed a

human-relevant concentration of PhIP.

The most striking result of this study is that the feeding

of rats with a supraphysiological concentration of folic acid

and a fat-rich diet leads to a significant increase in the

number of ACF per rat. These observations are the first to

be made in rats that have not concomitantly received a

known carcinogen. Since folic acid is not mutagenic (i.e.,

does not possess tumor initiating activity) and the fat-rich

diet did not include any genotoxins such as N-nitrosamines

and aflatoxins (see ‘‘Materials and methods’’) with tumor

initiating activity, the question as to the initiating stimulus

for the ACF remains unanswered. However, it has been

known for a number of years now that Fischer 344 rats

spontaneously develop a limited number of ACF through-

out their lifespan (Furukawa et al. 2002), and this has been

corroborated in this study: a low number of ACF were seen

in the group of rats fed a standard diet containing physio-

logical amounts of folic acid. Hence, the results obtained

herein could very well be the consequence of a supra-

physiological concentration of folic acid promoting the

growth of spontaneously initiated ACF and together with

previous reports demonstrating that the growth of chemi-

cally initiated ACF is significantly stimulated by supra-

physiological levels of folic acid (Reddy et al. 1996;

Wargovich et al. 1996, 2000; Lindzon et al. 2009) lend

support to the concept that folic acid supplementation, once

early preneoplastic lesions such ACF (Bird 1987; Tudek

et al. 1989) have developed, promotes carcinogenesis (Kim

1999; Choi and Mason 2002; Kim 2003, 2004).

As to the mechanisms involved in the tumor promoting

activity of folic acid, it has been proposed that increased

DNA methylation leads to the silencing of the expression

of regulatory or mismatch repair genes, which in turn may

provide a growth advantage to the initiated cells (Shen and

Issa 2002; Issa 2004; Wallace et al. 2010). Furthermore, it

has very recently been shown that multiple pathways

related to inflammation and immune response were

upregulated in rectosigmoid biopsies from volunteers

receiving supplemental folic acid (1 mg/day) for 8 weeks

(Protiva et al. 2011). In the case of the fat-rich diet, it has

been proposed that it upregulates the activity of cycloox-

ygenase-2 (Singh et al. 1997; Rao et al. 2001), thereby

leading to the enhanced production of prostaglandin E2,

which results in the activation of multiple intracellular

signaling pathways, including the transactivation of the

epidermal growth factor receptor and G-protein-mediated

activation of b-catenin/TCF-dependent transcription (Wu

et al. 2010).

In summary, the present study provides additional evi-

dence for a colon cancer promoting effect of folic acid

supplementation when rodents are fed the compound in

supraphysiological concentrations. While folic acid sup-

plementation in humans may prevent the development of a

colorectal tumor in those people without a preexisting

preneoplastic lesion in the colorectum, folic acid supple-

mentation in those people with an existing preneoplastic

colorectal lesion may support the development of a colo-

rectal tumor (Cole et al. 2007; Fife et al. 2011). Regardless

of the ongoing discussion on the pros and cons of folic acid

supplementation to prevent colorectal cancer and based on

the overwhelming body of evidence supporting the pro-

tective effect of periconceptional folic acid supplementa-

tion against neural tube defects, cleft palate, spina bifida,

and anencephaly, the advice for women planning a preg-

nancy to increase folic acid intake during the preconception

period to prevent the above-mentioned pregnancy out-

comes remains valid.

Acknowledgments This study was supported by a grant from the

Federal Institute for Risk Assessment in Germany (grant FK 3-1329-

303).

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