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Nutrition 28 (2012) 428–435

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Nutrition

journal homepage: www.nutr i t ionjrnl .com

Basic nutritional investigation

Potato fiber protects the small intestinal wall against the toxicinfluence of acrylamide

Piotr Dobrowolski Ph.D. a,*, Pauline Huet Ph.D. b, Patrik Karlsson Ph.D. c, Sune Eriksson Ph.D. d,Ewa Tomaszewska Ph.D. e, Antoni Gawron Prof. a, Stefan G. Pierzynowski Prof. b,f

aDepartment of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Lublin, PolandbDepartment of Biology, Lund University, Lund, Swedenc Eurofins Food & Agro Sweden AB, Lidköping, Swedend Er DevCo Consultants AB, Källby, SwedeneDepartment of Biochemistry and Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, PolandfDepartment of Medical Biology, Institute of Agricultural Medicine in Lublin, Lublin, Poland

a r t i c l e i n f o

Article history:Received 4 May 2011Accepted 4 October 2011

Keywords:Potato fiberDietary fiberAcrylamideSmall intestineHistomorphometryMouse

* Corresponding author. Tel.: þ48-507-132-520; faE-mail address: piotr.dobrowolski@umcs.lublin.pl

0899-9007/$ - see front matter � 2012 Elsevier Inc. Adoi:10.1016/j.nut.2011.10.002

a b s t r a c t

Objective: Acrylamide is a neurotoxic, genotoxic substance present in many commonly consumedfood products and has been shown to have carcinogenic effects in rodents. The protective effects (ifany) of potato fiber preparations, composed of cell wall material from potatoes, against the toxicinfluence of dietary acrylamide on the small intestinal wall were investigated.Methods: Male mice of the BALB/c strain were used in the study. Acrylamide was administered tothe mice in their drinking water (0.5 mg/kg of body weight per day) and one of two types of potatofiber preparations (heated or raw potato fiber preparation) was added to their feed (2% addition totheir feed). Histomorphometry of the small intestinal wall, hemoglobin adducts of acrylamide,animal weight, and feed and water consumption analyses were performed.Results: Acrylamide altered the morphology and histology of the small intestinal wall, decreasingproliferation, myenteron and submucosal thicknesses, villus length, fractal dimension, crypt depth,crypt number, and the small intestinal absorptive surface. Conversely, apoptosis, hemoglobinadduct levels, intensity of epithelium staining, enterocyte number, villus epithelial thickness, andcrypt width and parameters associated with nerve ganglia were increased. The two potato fiberpreparations that were used abolished the negative influences of acrylamide on the small intestinalwall and had no influence on the hemoglobin adduct levels of acrylamide.Conclusion: The negative impact of acrylamide on the histologic structure, regeneration, andinnervation of the small intestinal wall and the absorptive function of the small intestinal mucosacan be abolished by dietary potato fiber preparations.

� 2012 Elsevier Inc. All rights reserved.

Introduction

Many carcinogenic substances are present in food. In addi-tion, cooking and other food-processing methods can generatecarcinogenic from non-carcinogenic substances [1]. Thermaltreatment of certain amino acids (e.g., asparagine) particularly incombination with reducing sugars results in the formation ofacrylamide [2–5]. Acrylamide is a neurotoxic, genotoxicsubstance that has carcinogenic effects in rodents. Acrylamide isalso classified as a probable human carcinogen [6–10]. High

x: þ48-81-537-5901.(P. Dobrowolski).

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levels of acrylamide have been detected in commonly consumedfood items [1,5,11]. These data have motivated studies on the riskassessment of acrylamide in food [7,10,12]. According to Dybingand Sanner [13] and Dybing and Farmer [14], 6 of 10 000 indi-viduals may develop cancer as a result of ingesting acrylamidepresent in food products. In consequence, studies have beenconducted on lowering the level of acrylamide in processed food[15,16].

In addition to the above-mentioned substances, there arespecific proteins/peptides, e.g., digestive enzyme inhibitors, lec-tins, glyco-alkaloids, or dietary fiber, present in the diets, whichcan act as anticarcinogens [17–21]. Some functional food-activecomponents have been investigated and standardized, showing

Table 1Feed composition

Material Group content (g/kg)

NC/PC RP HP

Wheat 519.0 499.0 499.0Barley 100.0 100.0 100.0Oats 50.0 50.0 50.0Linseed meal 20.0 20.0 20.0Wheat bran 100.0 100.0 100.0Dry sugar beet pulp 80.0 80.0 80.0Fish meal LT999 40.0 40.0 40.0Casein sodium 30.0 30.0 30.0Concentrate protein whey PC6 30.0 30.0 30.0Phosphate-2-calcium 5.0 5.0 5.0Chalk fodder 15.0 15.0 15.0Salt fodder 3.0 3.0 3.0Premix LRM 8.0 8.0 8.0Raw potato fiber d 20.0 d

Heated potato fiber d d 20.0Total 1000.0 1000.0 1000.0

HP, heated potato fiber group, animals receiving a basal standard feed plus 2%heated potato fiber plus acrylamide 0.5 mg/kg of body weight per day in water;NC, negative control group, animals receiving a basal standard feed and noacrylamide in water; PC, positive control group, animals receiving a basal stan-dard feed plus acrylamide 0.5 mg/kg of body weight per day in water; RP, rawpotato fiber group, animals receiving a basal standard feed plus 2% non-heatedpotato fiber plus acrylamide 0.5 mg/kg of body weight per day in water

P. Dobrowolski et al. / Nutrition 28 (2012) 428–435 429

an ability to inhibit intestinal carcinogenesis in mice, rats, andhamsters and limiting carcinogenesis in several tissues andorgans such as the mammary glands, prostate glands, nervoussystem, and gastrointestinal tract [21–23]. Epidemiologic studiesthat have used soybean trypsin inhibitors, which also exist inpotato fiber preparations (potato trypsin inhibitor-1 and -2), asa dietary supplementation have shown a decreased occurrenceof breast, colon, nervous system, and prostate cancers in a pop-ulation consuming legumes and potatoes [20,24–26]. Thus, onecan speculate that legume diets containing specific proteins (e.g.,trypsin and amylase inhibitors) and potato lectins and fibersmight have an inhibitory effect on cancer development [17,21,27]. Food processing often involves heating, but trypsin inhibi-tors are thermally unstable and cooking partly or completelyeliminates their enzymatic activity in food [28,29]. With this inmind, we decided to investigate the effects of raw and heatedpotato fiber preparations, containing concentrated amounts ofenzyme inhibitors and dietary fiber, on the morphology of thesmall intestinal wall in mice.

The aim of the present study was to investigate whetheracrylamide has an influence on the structure of the small intes-tinal wall and if dietary supplementation with raw (non-heated)and heated potato fiber preparations would have a protectiveeffect against the toxic influence of acrylamide on the smallintestinal wall.

Materials and methods

Laboratory animals and experimental feed

The present study was reviewed and approved by the ethics committee ofLund University (application M140-03).

Forty 9-wk-old male mice of strain BALB/c, weighing approximately 20 geach, were used in the study. Mice were clinically healthy and housed in theirrespective groups in four separate cages (one cage per group, 10 mice in eachcage) under standard laboratory conditions (controlled temperature, humidity,and 12-h photoperiod; all experiments were carried out in the summer), withfree access to fresh water and solid food. Feed composition was based on thenutrient requirements of laboratory animals [30]. The feed was weighed weeklyand the feed consumption was calculated. The mice were randomly divided intofour groups according to the dietary supplementation: negative control group(NC)dmice received a basal standard feed and no acrylamide in water; positivecontrol group (PC)dmice received a basal standard feed and water with acryl-amide 0.5 mg/kg of body weight (BW) per day of acrylamide; raw potato fibergroup (RP)dmice received a basal standard feed with a 2% addition of non-heated potato fiber and water with acrylamide 0.5 mg/kg BW per day; heatedpotato fiber group (HP)dmice received a basal standard feed with a 2% additionof heated potato fiber and water with acrylamide 0.5 mg/kg BW per day. Micewere weighed monthly during the experimental period, which lasted for 90 d. Atthe end of the experimental period, all mice were euthanized, one by one, usingcarbon dioxide and dislocation of the spine. Blood and tissue samples werecollected from each mouse.

Feed and dietary fiber preparation composition

The experimental feed was provided by the Feeds and ConcentratesProduction Plant (Kcynia, Poland; Table 1). The potato fiber preparation (Povex),composed of cell wall material from potatoes, was produced by Lyckeby CulinarAB (Fjälkinge, Sweden). The Povex contained 82% carbohydrates (70% dietaryfiber and 12% starch and other small-molecule carbohydrates), 5% proteins, 0.4%fat, calcium, and phosphorus. Raw (non-heated) and heated potato fiber prepa-rations were used in the present study. The heating procedure was conducted indry air at a temperature of 160�C for 30 min.

Preparation and analysis of drinking water

Commercial acrylamide for electrophoresis in the form of powder (minimum99%; Sigma-Aldrich, Pozna�n, Poland) was used in the study. Diluted or powderedacrylamide was kept in a freezer at �20�C.

Water consumption during 24 h for the four groups of mice was measuredbefore the beginning of the experiment, and the data were used to calculate theamount of acrylamide administered, i.e., 0.5 mg/kg BW per day. The bottles were

filled with 600 mL of acrylamide 0.000625 g/L in a tap water solution and thewater was changed weekly. The water volume remaining in the bottles wasmeasured and tabulated to follow the consumption of acrylamide through thewater. There were no differences in water consumption among the groups. Threemilliliters of solution was sampled at the preparation of the acrylamide solutionand then again at the end of the week. These samples were stored at �20�C tocheck the stability of acrylamide in tap water. Analysis showed that the acryl-amide solution was stable for 1 wk.

Analysis of acrylamide content of feed and water

Samples of the control and experimental feeds and water were collected andthe acrylamide content was analyzed using a previously described method [31,32]. The homogenized samples from the feed preparation (normally approxi-mately 10 g) were extractedwith water (100mL) and the internal standard, [13C3]acrylamide, was added. The extract was analyzed by liquid chromatographictandem mass spectrometry (electrospray positive ionization - ESIþ) at ambienttemperature. The analysis of water was performed by a direct injection into theliquid chromatographic tandem mass spectrometer (ESIþ). All analyses wereperformed using the equipment described by Eriksson et al. [32].

Blood sampling and preparation

Blood samples were collected by cardiac puncture into heparinized tubes onice to induce hemolysis. The blood was separated into red blood cells and plasmaby centrifugation at 3000 rpm (Versatile Sigma 2-16P centrifuge, Sigma Labor-zentrifugen GmbH, Harz, Germany) for 5 min, and the red blood cell fractionwaswashed three times with 0.9% (w/v) saline (centrifuged after each washing) andstored at �20�C for hemoglobin adduct analysis.

Hemoglobin adduct concentration analysis

Hemoglobin adduct analysis was performed by liquid chromatographictandem mass spectrometry according to the method of Fennell et al. [33].Analyses were performed using the equipment described by Eriksson et al. [32].

Tissue collection and analysis

Small intestinal samples, from the pylorus to the cecum, were collected, andthe samples were weighed and the lengths were measured.

Two 10-mm-long segments of the small intestine from the jejunum (alwaysthe same place, 50% of total intestinal length) were taken from each mouse. Thetissues were fixed in 4% buffered formaldehyde (pH 7.0) for 24 h, dehydrated ingraded ethanol solutions, and embedded in paraffin. Twenty cross sections (with10-mm interval after each five-slice section) 5 mm thick were cut with a micro-tome (Microm HM 360, Microm, Walldorf, Germany) from every sample of the

P. Dobrowolski et al. / Nutrition 28 (2012) 428–435430

small intestine. Two common methods of staining were used: hematoxylin andeosin and Hoechst eosin [34].

Microscopic (two-dimensional) images were collected using a confocalmicroscope (Axiovert 200M, equipped with an LSM Pascal 5 scanning head; CarlZeiss, Jena, Germany) with an argon laser wave length of 514 nm, and the imageswere combined from two channels: laser scans and the Nomarski technique. Inaddition, a light and fluorescent microscope (excitation wavelength 450–490nm; Nikon Eclipse E-800, Nikon, Tokyo, Japan) equipped with a Nikon D70 digitalcamera was used. Objective magnifications of 4�, 10�, 20�, 40�, 63�, and 100�were used to show the different intestinal structures and to collect images of theexamined tissues from each specimen for further analysis.

The structure of the small intestinal wall was examined under microscopicobservation and with the use of graphic analysis software (ImageJ 1.44d, NationalInstitutes of Health, Bethesda, MD, USA; available at: http://rsb.info.nih.gov/ij/index.html).

Histomorphometric analysis of small intestinal wall

The morphometric variables analyzed included the number of mitosescalculated per millimeter of crypt epithelium. Mitoses were identified from lateprophase to telophase in the epithelium of 30 well-defined and verticallyoriented crypts (from 10 slices from every mouse). Mitoses were countedclockwise from the coincidental start point on the small intestinal cross section;the criteria for recognizing mitotic figures were those outlined by van Diest et al.[35]. Apoptotic cells were counted per square millimeter of tissue (in Hoechsteosin–stained sections, apoptotic cells per approximately 2000 cells); the criteriafor recognizing apoptotic cells were those outlined by Lizard et al. [36] andMajnoand Joris [37]. In addition, histograms of fluorescence distribution were analyzedto assess the homogeneity of the DNA condensation in fragmented or condensednuclei, in the case of an uncertain assessment of apoptotic cells. The perimeter ofthe small intestinal cross section, the fractal dimension of the small intestinalmucosa (according to the box-counting method [38]), mucosa, submucosa, andmyenteron thickness were measured. The crypt depth (defined as the depth ofthe invagination between adjacent villi, from the bottom of crypt to the base ofthe villus), crypt width (measured in the middle of the crypt depth), number ofcrypts (active: showing mitoses, having an open internal space and access to theintestinal lumen; inactive: showing no mitoses and having a closed internalspace; total: active plus inactive crypts), villus fractal dimension (according to thebox-counting method [38]), villus height (from the tip of the villus to the villus–crypt junction), villus width (measured in the middle of the villus height),number of villi per millimeter of mucosa, and number of enterocytes per 100 mmof villus epithelium were measured. The intensity of villus epithelium staining(measured as an 8-bit gray value, which showed an inversely proportional cor-relationdthe lower the result, the stronger the tissue staining), villus epitheliumthickness (cell length measured as the distance from brush-border membrane tothe basolateral membrane), and small intestinal absorptive surface [39] were alsodetermined. Only vertically oriented villi and crypts were measured.

Immunohistochemistry analysis of small intestinal wall

Immunohistochemical staining with monoclonal rat anti-mouse antibodiesagainst Ki-67 [40] and rabbit anti-mouse polyclonal to 200-kD neurofilamentantibodies were performed to identify proliferating cells and to localize neuronalcells, respectively. Antigen retrieval of formalin-fixed, paraffin-embedded tissuesections of the small intestine was achieved by microwave (700 W) heating(three times for 5 min) in citrate buffer (pH 6) followed by deparaffinizing andrehydration [41]. Endogenous peroxidase activity was blocked subsequently witha 3% solution of hydrogenperoxide inmethanol (1:1) for 30min. After blocking innormal serum, sections were incubated with monoclonal rat anti-mouse anti-bodies against Ki-67 (dilution 1:50; DacoCytomation, Glostrup, Denmark) orrabbit polyclonal to 200-kD neurofilament antibodies (dilution 1:500; Abcam,Cambridge, UK) overnight at 4�C. The sections were then incubated with bio-tinylated anti-rat immunoglobulin (dilution 1:200; DacoCytomation) withstreptavidin/horseradish peroxidase (dilution 1:300; DacoCytomation) or bio-tinylated goat polyclonal to rabbit immunoglobulin (dilution 1:200; Abcam) withstreptavidin/horseradish peroxidase (dilution 1:300; DacoCytomation), respec-tively. Then, the sections were developed in 3030-diaminobenzidine tetrahydro-chloride (DacoCytomation) as a chromogene. Counterstaining with hematoxylinwas used. Negative control sections from each mouse received identical prepa-rations for immunohistochemical staining, except that the primary antibodieswere omitted.

Microscopic images of these immunohistochemistry reactions were sub-jected to further analysis, and the variables analyzed included the number ofproliferating cells, the area of proliferation, and the intensity of the reaction(measured as an 8-bit gray value that showed an inversely proportional corre-lationdthe lower the result, the stronger the tissue staining) for Ki-67 and thecross-sectional area of the nerve ganglion, the intensity of the neurofilamentimmunohistochemistry reaction (measured as an 8-bit gray value that showed an

inversely proportional correlation), the circularity of the nerve ganglion, and theneurofilament integrated density for neurofilament detection.

Statistical analysis of data

All results are expressed as mean � standard deviation. Differences betweenmeans were analyzed using a nested analysis of variance and the Tukey post hoctest as a correction for multiple comparisons. The normal distribution of datawasexamined using the Shapiro-Wilk test, and the equality of variance was testedusing the Brown-Forsythe test. When there was a lack of normal distributionand/or unequal variance of data, the Friedman analysis of variance was used toanalyze the differences between means. P < 0.05 was considered statisticallysignificant. All statistical analyseswere carried out using STATISTICA 8.0 (StatSoft,Inc.; available at: www.statsoft.com).

Results

Analyses of body mass, water consumption, and the length,perimeter, and mass of the small intestine showed no significantdifferences among the groups (data not shown). There were nodeaths during the experiment.

Feed consumption

Feed consumption of mice from the RP group (142.1 �14.4 g/kg BW per day) was significantly lower (P ¼ 0.0038) than in theHP group (161.8 � 16.0 g/kg BW per day). There were nosignificant differences between the NC (153.7 � 9.2 g/kg BW perday) and the PC (154.8 � 12.5 g/kg BW per day, P ¼ 0.99) orbetween the control and the other groups (P > 0.05).

Acrylamide content in feed and hemoglobin adduct level

Aminimal amount of acrylamidewas detected by the analysisof feed samples: 8 mg/kg for the basal standard feed, 11 mg/kg forthe feed with the 2% addition of raw (non-heated) potato fiber,and 13 mg/kg for the feed with the 2% addition of heated potatofiber. The calculated amounts of acrylamide taken with the feedwere approximately 1.2 mg/kg BW per day for the NC and PCgroups, 1.5 mg/kg BW per day for the RP group, and 2 mg/kg BWper day for the HP group.

Hemoglobin adducts were detected in the amounts of 0.08 �0.01, 0.10 � 0.00, and 0.09 � 0.02 pmol/mg of globin for the PC,RP, and HP groups, respectively. The amount of hemoglobinadducts in the NC group was below the detection level (<0.01 �0.00). Mice receiving acrylamide showed a significantlyincreased level of hemoglobin adducts compared with the NCgroup (PC, P ¼ 0.0051; RP, P ¼ 0.0009; HP, P ¼ 0.0017). Therewere no significant differences among the PC, RP, and HP groups(P ¼ 0.28, 0.68, and 0.86, respectively).

Cell cycle, renewal, and apoptosis in small intestine

Almost all cells in the active stages of the cell cycle (positiveKi-67 reaction) were observed in the small intestinal crypt area(Fig. 1). The number of proliferating cells was significantlydecreased by the acrylamide; however, some improvement wasobserved in mice receiving the raw potato fiber and a significantimprovement was observed in mice receiving the heated potatofiber (Table 2). Nevertheless, the area of proliferation and theintensity of the Ki-67 antigen reaction in mice receiving the rawpotato fiber were significantly decreased compared with thenegative control. Moreover, these two last parameters were notchanged by acrylamide (Table 2).

The mitotic number in crypt epithelium was decreased byacrylamide. Mice receiving acrylamide and the heated potato

Fig. 1. The influence of raw and heated potato fiber preparations on the nerve ganglia and the regeneration of the small intestine in mice treated with acrylamide in drinkingwater (0.5 mg/kg of body weight per day). Immunohistochemical reactions using 200-kD neurofilament and Ki-67 antibodies were carried out on formaldehyde-fixedsections. Animals receiving acrylamide showed less intense staining of the nerve ganglia (arrows); nevertheless, the PR and HP groups showed a protective effect ofpotato fiber as indicated by the stronger staining compared with the PC group. Proliferating cells are visualized as brown-colored structures in the Ki-67 reaction (right).Animals receiving only acrylamide showed fewer proliferating cells than the other mice. The largest number of cells with the most intense reaction was present in the RPgroup. HP, animals receiving a basal standard feed plus 2% heated potato fiber plus acrylamide 0.5 mg/kg of body weight per day in water; NC, animals receiving a basalstandard feed and no acrylamide in water; PC, animals receiving a basal standard feed plus acrylamide 0.5 mg/kg of body weight per day in water; RP, animals receivinga basal standard feed plus 2% non-heated potato fiber plus acrylamide 0.5 mg/kg of body weight per day in water.

P. Dobrowolski et al. / Nutrition 28 (2012) 428–435 431

fiber showed a similar decrease, but not as great as that observedin the mice receiving acrylamide alone. Conversely, the mitoticnumber was increased by the raw potato fiber above the levelobserved in the negative control group (Table 2).

Apoptotic cell number was significantly increased by acryl-amide, although not in mice receiving the raw or heated potatofiber (Table 2).

Histomorphometry of small intestinal wall

The myenteron thickness, the intensity of epithelium staining(higher values indicate less intensive dye absorption), villuslength and fractal dimension, crypt depth, active, inactive, andtotal crypt numbers, and the small intestinal absorptive surfacewere significantly decreased by acrylamide (Table 3). Conversely,

Table 2Effects of raw and heated potato fiber preparations on regeneration of the small intestine in mice receiving acrylamide in drinking water (0.5 mg/kg of body weight perday)

Parameter NC PC RP HP

Proliferating cells/mm2 tissue 1929 � 619a 1260 � 588b 1772 � 406a,b 1971 � 606a

Area of proliferation (mm2)/mm2 tissue 0.11 � 0.06a 0.08 � 0.06a,b 0.04 � 0.05b 0.06 � 0.04a,b

Intensity of Ki-67 antigen reaction (8-bit gray scale)* 97.9 � 23.2a 97.0 � 15.6a 81.1 � 16.2b 97.7 � 10.2a

Mitotic number/mm crypt epithelium 1.55 � 0.09a 1.26 � 0.04b 1.76 � 0.13a 1.31 � 0.04b

Apoptotic cells in small intestine/mm2 tissue 0.63 � 0.03a 2.54 � 0.64b 0.85 � 0.63a 1.26 � 0.74a

Data are expressed as mean � SD (n ¼ 10/group). Superscript letters indicate significant differences among groups at P < 0.05HP, heated potato fiber group, animals receiving a basal standard feed plus 2% heated potato fiber plus acrylamide 0.5 mg/kg of body weight per day in water; NC,negative control group, animals receiving a basal standard feed and no acrylamide in water; PC, positive control group, animals receiving a basal standard feed plusacrylamide 0.5 mg/kg of body weight per day in water; RP, raw potato fiber group, animals receiving a basal standard feed plus 2% non-heated potato fiber plusacrylamide 0.5 mg/kg of body weight per day in water

* Analysis of the 8-bit gray scale shows an inversely proportional correlation, i.e., the lower the value, the stronger the tissue staining.

P. Dobrowolski et al. / Nutrition 28 (2012) 428–435432

the enterocyte number in the villus epithelium, villus epitheliumthickness, and crypt width were increased by acrylamide (Table3). For these parameters (except the intensity of epitheliumstaining), the identified action of acrylamide was reversed by theadministration of the raw and/or heated potato fiber preparation(Table 3). For the submucosal and mucosal thicknesses, mucosafractal dimension, and villus thickness and number, the influenceof acrylamide was not significant compared with the NC mice,although the two potato fiber preparations showed oppositeinfluences (except the effect of the raw potato fiber on submu-cosa and villus thicknesses; Table 3).

The intensity of neurofilament staining (higher valuesindicate less intensive dye absorption), circularity of the nerveganglia, and the neurofilament integrated density weremarkedly increased by acrylamide (Fig. 1). Moreover, thecross-sectional area of the nerve ganglion was increased byacrylamide, but this was not significant (Table 4). The parametersof the nerve ganglion changes induced by acrylamide weresignificantly reversed by the administration of the heated potatofiber. The raw potato fiber showed a less prominent effect, andonly the circularity of the nerve ganglion was significantly low-ered by this preparation compared with the PC group (Table 4).

Table 3Effects of raw and heated potato fiber preparations on histomorphometry of the small iper day)

Parameter NC

Myenteron thickness (mm) 27.8 � 5.7a

Submucosa thickness (mm) 19.5 � 5.4a

Mucosa thickness (mm) 493.4 � 43.3a,b

Mucosa fractal dimension (D) 1.27 � 0.03a,b

Intensity of epithelium staining (8-bit gray scale)* 77.0 � 25.2a

Enterocytes/100 mm of villus 14.2 � 2.3a

Villus epithelium thickness (mm) 23.8 � 2.6a

Villus fractal dimension (D) 1.68 � 0.08a

Villus length (mm) 422.81 � 60.2a

Villus thickness (mm) 70.4 � 10.5a,b

Villi/mm 10.0 � 2.1a

Crypt depth (mm) 78.5 � 12.6a

Crypt width (mm) 29.7 � 4.3a

Active crypts/mm 8.0 � 3.7a

Inactive crypts/mm 19.7 � 4.7a

Total crypts/mm 27.7 � 6.7a

Small intestinal absorptive surface (mm2) 12.5 � 2.0a

Data are expressed as mean � SD (n ¼ 10/group). Superscript letters indicate significHP, heated potato fiber group, animals receiving a basal standard feed plus 2% heatenegative control group, animals receiving a basal standard feed and no acrylamide inacrylamide 0.5 mg/kg of body weight per day in water; RP, raw potato fiber groupacrylamide 0.5 mg/kg of body weight per day in water

* Analysis of the 8-bit gray scale shows an inversely proportional correlation, i.e., t

Discussion

The toxic influence of acrylamide on the small intestinal wallof mice was investigated. Raw (non-heated) and heated potatofiber preparations were used to explore their possible protectiveeffects against the toxic influence of acrylamide on the smallintestinal wall.

A daily dose of acrylamide 0.5 mg/kg BW was chosen for thepresent study because it was one of the lowest doses shown inprevious studies to induce tumors in rodents and was one of thedoses tested in animals that was closest to the daily intake ofacrylamide in humans [10,14]. The 2% addition of potato fiberwasused in the present study because it has been previously shownto possess protective properties with respect to the digestivetract of weaned piglets and thus a potentially beneficial agent(personal communication, Stefan G. Pierzynowski 2001) [42,43].

Although rodents are more sensitive to acrylamide exposurethan humans (in neurotoxicity and genotoxicity), we used themouse model because of the relatively short span of the exper-iment and the relatively small dose of acrylamide required.Considering our dose of acrylamide and its ability to inducecancer formation, we chose to go in this direction to make our

ntestine inmice receiving acrylamide in drinkingwater (0.5mg/kg of body weight

PC RP HP

20.8 � 5.6b 28.6 � 8.2a 29.3 � 4.1a

18.0 � 4.2a,b 14.8 � 3.1b 19.5 � 2.4a

471.1 � 92.9a 506.5 � 69.8a,b 540.2 � 65.3b

1.22 � 0.03a 1.28 � 0.01b 1.27 � 0.03a,b

86.9 � 19.8b 84.1 � 24.5b 84.4 � 25.6b

17.8 � 2.2b 14.7 � 1.4a 14.2 � 1.9a

25.6 � 2.9b 24.7 � 3.1c 23.8 � 3.3a

1.63 � 0.09b 1.69 � 0.08a 1.67 � 0.06a,b

393.7 � 77.1b 412.0 � 63.9a,b 449.2 � 54.6c

73.8 � 14.8a 67.6 � 9.3b 70.0 � 11.7a,b

9.9 � 1.0a 12.1 � 2.9b 11.7 � 2.4a,b

63.5 � 9.4b 69.4 � 10.6b 80.4 � 10.3a

33.9 � 4.6b 28.8 � 5.0ac 27.7 � 3.5c

3.2 � 2.0b 2.5 � 2.1b 7.1 � 2.7a

16.1 � 3.8b 21.0 � 3.7a 19.1 � 3.2a,b

19.4 � 3.4b 23.6 � 4.3c 26.3 � 4.0ac

10.6 � 2.3b 12.6 � 2.0a 13.8 � 2.0c

ant differences among groups at P < 0.05d potato fiber plus acrylamide 0.5 mg/kg of body weight per day in water; NC,water; PC, positive control group, animals receiving a basal standard feed plus

, animals receiving a basal standard feed plus 2% non-heated potato fiber plus

he lower the value, the stronger the tissue staining.

Table 4Effects of raw and heated potato fiber preparations on the nervous system of the small intestine in mice receiving acrylamide in drinking water (0.5 mg/kg of bodyweight per day)

Parameter NC PC RP HP

Cross-sectional area of nerve ganglion (mm2) 128.8 � 87.9a,b 181.5 � 108.9a 136.9 � 91.7a,b 110.1 � 83.8b

Intensity of neurofilament staining (8-bit gray scale)* 147.6 � 17.2a 167.5 � 12.2b 161.9 � 14.6b 148.1 � 17.5a

Circularity of nerve ganglion 0.37 � 0.15a 0.59 � 0.15b 0.43 � 0.17a 0.45 � 0.21a

Neurofilament integrated density 55.1 � 38.6a 88.0 � 53.3b 64.5 � 44.6a,b 47.8 � 37.5a

Data are expressed as mean � SD (n ¼ 10/group). Superscript letters indicate significant differences among groups at P < 0.05HP, heated potato fiber group, animals receiving a basal standard feed plus 2% heated potato fiber plus acrylamide 0.5 mg/kg of body weight per day in water; NC,negative control group, animals receiving a basal standard feed and no acrylamide in water; PC, positive control group, animals receiving a basal standard feed plusacrylamide 0.5 mg/kg of body weight per day in water; RP, raw potato fiber group, animals receiving a basal standard feed plus 2% non-heated potato fiber plusacrylamide 0.5 mg/kg of body weight per day in water

* Analysis of the 8-bit gray scale shows an inversely proportional correlation, i.e., the lower the value, the stronger the tissue staining.

P. Dobrowolski et al. / Nutrition 28 (2012) 428–435 433

study relevant to human physiology. In cancer tests, mice weremore sensitive to tumor induction per unit of absorbed lifetimedose of acrylamide comparedwith rats [8,44]. Themouse is moreeffective than the rat in metabolizing acrylamide to glycinamide[45,46]. The multiplicative cancer risk model for acrylamideimplies that the increase in cancer risk of a genotoxic chemical isproportional to the in vivo dose of the genotoxic compound andthe background tumor frequency [47]. Acrylamide monomerspass the intestine cells monolayer by passive diffusion and,therefore, a high bioavailability can be expected [48]. Acrylamideforms adducts with hemoglobin, which can be measured in theblood [11,49]. Measurement of these adducts reflect the effectiveacrylamide doses that pass through the gastrointestinal tractbarrier [11]. In our study, all mice receiving acrylamide hadincreased levels of hemoglobin adducts of acrylamide, irre-spective of whether they received the raw or heated potato fiberpreparation. The levels of hemoglobin adducts of acrylamidewere not influenced by the feed supplementation, whereas locallesions and toxic effects were observed in the examined struc-tures of the small intestinal wall. In our study, the observedeffects of acrylamide may suggest that the potato fiber prepa-rations used did not decrease the bioavailability of acrylamide,although a decreased bioavailability of acrylamide by dietaryfiber has previously been suggested by Woo et al. [50]. Althoughacrylamide had no effect on the consumption of feed or water,body weight, or mortality in our study, this is contrary toa previous study that showed that the long-term effects ofacrylamide included a decreased body weight and increasedmortality [51]. Our findings showed that acrylamide hada negative influence on small intestinal regeneration and thehistomorphometric parameters associated with the physiologicfunctions of this organ (Tables 2 and 3, Fig. 1) and on the smallintestinal nervous system (Table 4, Fig. 1), which in turn may bea predominant source of small intestinal lesions, as shownpreviously [52]. However, the raw and heated potato fiberpreparations clearly decreased the impact of acrylamide on themorphologic parameters of the small intestinal wall, on prolif-eration, and, most importantly, on the absorptive surface of thesmall intestine of mice treated with acrylamide (Tables 2 and 3).The feed supplementation with the potato fiber preparationspreserved the apoptotic level and the physiologic mitoticnumber in the most metabolically active region of the intestinalmucosadthe crypt region, which in turn is one of the mostmetabolically active regions in the entire body.

Before the start of the experiment, the potential enzymaticactivity of the raw potato fiber preparation was expected to besomewhat similar to that of the trypsin inhibitors fromsoybeans [26]. Heating of the potato fiber preparation for 30min at 160�C in the third preparation was done to denature theproteins and thus stop all potential enzymatic activity [28,29].

Moreover, the time and temperature used in the experiment areoften used in cooking in the ordinary household kitchen. It isworth emphasizing that the maximum intensity of synthesis ofacrylamide occurs from 160�C to 180�C [2]. Nevertheless, wefound a positive, protective effect of the raw and heated potatofiber preparations (Tables 2–4, Fig. 1), indicating a differentmechanism (active substances) behind these diverse protectiveactions of the normal and heat-treated potato fiber prepara-tions. Moreover, in the standard feed, more than 4% dietary fiberalready exists; therefore, the 2% addition of potato fiber (heatedor raw) confirmed the activity of the two potato fiber prepa-rations used. It must be stressed that the heated potato fiberpreparation showed an even stronger preventative effectagainst the changes of the examined parameters of the smallintestinal wall caused by acrylamide (in 17 examined parame-ters) than the raw fiber preparation (in 12 examined parame-ters; Tables 2–4).

There are several limitations to the present study. First, thestudy did not include a group that received potato fiber withoutacrylamide. However, it is not very likely that people will beconsuming food without acrylamide in an ordinary daily diet,especially in industrialized countries, because most meals arecooked. Moreover, dietary fiber was already present in thecontrol group. Most animal feeds contain acrylamide from theheating that occurs during the preparation, so we would haveone more variable (on a different level) that was already presentin the study. Our goal was to study whether acrylamide had aninfluence on the structure of the small intestinal wall, and, if so,whether there was a possible substance that could decrease orprevent this effect. The interactions between acrylamide andpotato fiber would be interesting in such situations. The influ-ence of dietary fiber on the small intestine is known. Second, theglycinamide adduct levels and the kinetics of acrylamide uptakeinto the blood were not measured. However, our goal was todetermine if there were any histologic changes in the smallintestinal wall caused by dietary acrylamide supplementation,although the analysis described earlier would have been usefulin explaining themechanism and ratio of the effect of acrylamideon small intestinal physiology. Third, the activity of the enzymeinhibitors from the potato fiber preparations was not measured.However, there are several studies on this subject stating thisactivity [18,25,26]. It would be interesting to determine whetherthere is any specific property or activity associated with thecomponents of protein or the other components of the potatofiber preparations that gives it the protective properties againstacrylamide toxicity.

The present study has its advantages. This is the first study toshow the effects of acrylamide on the histologic structure of thesmall intestinal wall. However, because of the low dose ofacrylamide used, the relatively short experimental period, and

P. Dobrowolski et al. / Nutrition 28 (2012) 428–435434

no external signs of toxicity or neurologic changes, the observedsignificant impact of acrylamide was not originally expected.Moreover, our study showed different and positive effects of theraw and heated potato fiber preparations, which, according toprevious studies showing the anticarcinogenic effects of enzymeinhibitors in food products, definitely were not expected for theheated potato fiber. Furthermore, to our knowledge, there are noother studies on other dietary fibers that have a protective effectagainst the toxic influence of acrylamide. Moreover, the study byWoo et al. [50] showed a lack of the protective effects of dietaryfiber or chlorophyllin against acrylamide toxicity. Our researchrevealed different effects of the raw and heated potato fiberpreparations, which may suggest specific beneficial influences ofthis dietary fiber on experimentally induced acrylamide toxicity;however, the mechanism of action remains unknown andrequires further study. Although heating eliminated most of theknown thermolabile elements, e.g., enzyme inhibitors existing inthe raw potato fiber preparation, surprisingly, the heated potatofiber exhibited even stronger positive effects. Accordingly, theheating process probably results in the formation of newsubstances that protect the small intestinal wall against theinfluence of acrylamide. Further studies are needed for theidentification of these new substances and to elucidate theirpossible beneficial effects on the physiology of the gastrointes-tinal tract.

Conclusion

Our results showed the negative impact of acrylamide on thehistologic structure, regeneration, and innervations of the smallintestinal wall and, indirectly, on the absorptive function of thesmall intestinal mucosa. These effects were mostly abolished bythe use of the dietary fiber preparations.

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