https://doi.org/10.52973/rcfcv-e34500

Received: 06/08/2024 Accepted: 28/10/2024 Published: 31/12/2024

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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34500

ABSTRACT

This study was aimed at determining the effects of wheat gluten,

corn gluten and soybean meal, incorporated into feed as protein

sources, on the hepatic tissue of rats, based on the investigation of

histopathological parameters (degeneration, inammation, biliary

hyperplasia and fat droplets), immunohistochemical parameters

(transglutaminase, gliadin, IgA, IgG, CD4 and CD8), and the serum

levels of hepatic enzymes [(aspartate aminotransferase (AST),

alanine transaminase (ALT), alkaline phosphatase (ALP), lactate

dehydrogenase (LDH)]. Three groups, referred to as Wheat Group,

Group Corn and Group Soybean, were established, and were given

high levels of wheat gluten, corn gluten and soybean meal as protein

sources in the feed, respectively. The study material comprised

forty–eight 20–day–old female Sprague Dawley rats. In the group,

which received dietary wheat gluten, the hepatic tissue presented

with numerically higher histopathological and immunohistochemical

parameters on day 45 of the study, and numerically higher

histopathological parameters on day 165 of the study (P>0.05). On

day 165 of the study, when compared to Groups Corn and Soybean,

Group Wheat displayed a signicantly different level of sensitivity to

immunohistochemical parameters (transglutaminase, gliadin, IgA, IgG,

CD4 and CD8). It was determined that gluten signicantly reduced the

activity of the liver enzymes LDH and AST. In result, it was ascertained

that rats, not carrying the genes HLA–DQ2 and DQ8, when fed on a

ration incorporated with a high level of wheat gluten, displayed slightly

affected hepatic tissue histopathological parameters and signicantly

increased immunohistochemical parameters (transglutaminase,

gliadin, IgA, IgG, CD4 and CD8).

Key words: Rat; gluten; wheat; corn; immunohistochemical

RESUMEN

Este estudio tuvo como objetivo determinar los efectos del gluten

de trigo, gluten de maíz y harina de soya, incorporados al alimento

como fuentes de proteína, sobre el tejido hepático de ratas, con base

en la investigación de parámetros histopatológicos (degeneración,

inflamación, hiperplasia biliar y gotas de grasa), parámetros

inmunohistoquímicos (transglutaminasa, gliadina, IgA, IgG, CD4

y CD8), y los niveles séricos de enzimas hepáticas [(aspartato

aminotransferasa (AST), alanina transaminasa (ALT), fosfatasa alcalina

(ALP), lactato deshidrogenasa (LDH)]. Se establecieron tres grupos,

denominados Grupo Trigo, Grupo Maíz y Grupo Soja, a los que se les

suministró en el alimento altos niveles de gluten de trigo, gluten de

maíz y harina de soya como fuentes de proteína, respectivamente.

El material de estudio estuvo compuesto por cuarenta y ocho ratas

Sprague Dawley hembras de 20 días de edad. En el grupo que recibió

gluten de trigo en la dieta, el tejido hepático presentó parámetros

histopatológicos e inmunohistoquímicos numéricamente más altos

el día 45 del estudio, y parámetros histopatológicos numéricamente

más altos el día 165 del estudio (P>0,05). El día 165 del estudio, en

comparación con los grupos Maíz y Soja, el Grupo Trigo mostró un

nivel signicativamente diferente de sensibilidad a los parámetros

inmunohistoquímicos (transglutaminasa, gliadina, IgA, IgG, CD4 y

CD8). Se determinó que el gluten redujo signicativamente la actividad

de las enzimas hepáticas LDH y AST. Como resultado, se comprobó

que las ratas no portadoras de los genes HLA–DQ2 y DQ8, cuando

fueron alimentadas con una ración incorporada con un alto nivel de

gluten de trigo, mostraron parámetros histopatológicos del tejido

hepático ligeramente afectados y parámetros inmunohistoquímicos

signicativamente aumentados (transglutaminasa, gliadina, IgA,

IgG, CD4 y CD8).

Palabras clave: Rata; gluten; trigo; maíz; inmunohistoquímica

Effects of experimental wheat and corn gluten on liver tissue in

rats: biochemical, histopathological, immunohistochemical and

immunouorescence methods

Efectos del gluten de trigo y maíz experimental sobre el tejido hepático de ratas: métodos

bioquímicos, histopatológicos, inmunohistoquímicos y de inmunouorescencia

Kübra Asena Terim Kapakin

, Recep Gümüş

* , İsmail Bolat

, Esra Manavoğlu Kirman

, Aybüke İmik

Ataturk University, Faculty of Veterinary Medicine, Department of Veterinary Pathology. Erzurum, Türkiye.

Sivas Cumhuriyet University, Faculty of Veterinary Medicine, Department of Animal Nutrition and Nutritional Disorders. Sivas, Türkiye.

Faculty of Veterinary Medicine, Faculty of Health Sciences, Department of Nutrition and Dietetics. Selçuk University, Konya, Türkiye.

*Corresponding author: rgumus@cumhuriyet.edu.tr

TABLE I

Ingredients and nutrient composition of rat diet in the study

Ingredients, %

Groups

Wheat Corn Soybean

Wheat bran 1.8 3.5 3.24

Oat, %11 CP 68 64 62.11

Sunower meal, % 28 CP 13 13 6

Corn gluten meal, % 62 CP - 17 -

Wheat gluten meal, % 75 CP 24.85 - -

Soybean meal, % 51 CP - - 24.85

Animal fat 2.2 1.5 2.8

Vitamin-mineral premix* 1 1 1

Nutrient composition

Crude protein, % 22 22 22

Metabolisable energy, (kcal·kg

-1

) 2,599 2,657 2,598

Ca, % 0.15 0.11 0.14

Methionine + cysteine, % 0.66 0.83 0.68

Lysine, % 1.17 0.63 1.15

*The vitamin-mineral premix provides the following (per kg): vitamin A 6,000,000 IU;

vitamin D3 800,000 IU; vitamin E 8,000 mg; vitamin K3 2,000 mg; vitamin B1 1,200 mg;

vitamin B2 3,000 mg; vitamin B6 2,000 mg; vitamin B12 8 mg; niacin 10,000 mg; folic

acid 400 mg; d-biotin 20 mg; choline chloride 160,000 mg; manganese 32,000 mg; iron

16,000 mg; zinc 24,000 mg; copper 2,000 mg; iodine 800 mg; cobalt 200 mg; selenium

60 mg; Cal-D-Pan. 4,000 mg; antioxidant 4,000 mg. CP: Crude protein

Effects of experimental wheat and corn gluten on liver tissue in rats / Terim Kapakin et al. _________________________________________

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INTRODUCTION

Wheat (Triticum aestivum) and corn (Zea mays) are two main cereal

grains, which are most commonly produced and consumed Worldwide

[1]. The main structural constituents of these grains are starch,

cellulose and gluten. The term gluten refers to a complex mixture

of proteins, which is found in grains such as wheat, corn, barley

(Hordeum vulgare), rye (Lolium multiorum) and oat (Avena sativa),

and remains after these grains are processed and washed of their

starch. Nearly 5.4% of wheat grain [2] and 3–3.55% of corn grain is

made of gluten [3].

Normally, gluten contains proteins that are readily digested in the

human intestines [4]. On the other hand, individuals not carrying the

genes HLA–DQ2 and DQ8 are not capable of digesting these proteins.

The consumption of gluten–containing diets by these persons may

lead to multiple health problems. These health problems include

coeliac disease, gluten intolerance, non–coeliac gluten sensitivity,

wheat allergy, and dermatitis herpetiformis [5, 6]. The sensitivity of

tissues to gluten is determined in view of histopathological ndings

and immunohistochemical analyses [7].

In the present study, healthy rats were provided with gluten–rich

feed for a long–term period and the sensitivity of their hepatic tissue

to gluten was investigated. Histopathological alterations in the

hepatic tissue were assessed on the basis of several parameters,

including degeneration, inammation, biliary hyperplasia, and fat

droplets, whilst immunohistochemical analyses were performed for

the measurement of transglutaminase, gliadin, IgA, IgG, CD4 and CD8

levels. Also, AST, ALT, ALP and LDH levels, which are liver enzymes,

were investigated in serum tissue.

MATERIALS AND METHODS

Animal material, experimental groups, and feed

In this study, 48 healthy female Sprague–Dawley rats (Rattus

norvegicus), 20 days (d) old, were raised until they reached 185 d of

age (the trial period lasted 165 d) by being fed experimental diets. The

rations given to the study groups were isonitrogenous and isocaloric

(TABLE I). Three study groups were established, named Group Wheat,

Group Corn, and Group Soybean, each receiving dietary wheat gluten,

corn gluten, and soybean meal, respectively. Throughout the study, the

animals were fed for 165 d, with feed and water available ad libitum,

and were maintained at a comfort temperature of 22°C.

In the present study, healthy rats, confirmed not to carry the

genes HLA–DQ2 and DQ8, were fed on rations supplemented with

high levels of wheat gluten and corn gluten for a period of 165 d, with

to investigate the effects of gluten and the sensitivity displayed by

hepatic tissue to gluten. The effects of gluten on hepatic tissue were

determined based on the measurement of histopathological and

immunohistochemical parameters and enzyme activity in hepatic

tissue samples on d 45 and 165 of the study.

Pathological examination

Rats were sacriced under anesthesia at the average ages of 65 and

185 d, and tissue samples were collected. Liver tissue samples were

obtained for histopathological and immunohistochemical analysis, and

lesions were scored semi–quantitatively based on the microscopic

(Olympus BX51, Japan) examination of 10 different areas at 40×

magnication. Histopathologically, Degeneration, Inammation,

Biliary hyperplasia and Fat drop scoring were performed in liver

tissues. Immunohistochemically, Transglutaminase, Gliadin, IgA,

IgG, CD4 and CD8 expression levels were scored. The scoring was

as follows: 0 (negative), +1 (slight), +2 (moderate), +3 (severe), and

+4 (very severe) [8].

Histopathological examination

For histopathological examination, tissue specimens were initially

xed in 10% buffered formalin for 48–72 hours and then rinsed under

running water for 6–8 hours. The specimens were then processed

using a routine tissue processing method, which involved passing

them through graded alcohols (70°, 80°, 90°, 96°, and 100°) and xylene

series. After embedding in paran, 4 μm thick sections were cut from

the paran blocks and mounted on glass slides. The histopathological

sections were stained with hematoxylin–eosin (HE) [9, 10] and were

examined and imaged using an Olympus BX52 1 light microscope

(Japan) with a DP72 camera system, at the Pathology Department

Laboratory of Atatürk University, Faculty of Veterinary Medicine.

Immunohistochemical examination

All sections mounted on poly–L–lysine–coated adhesive glass slides

for immunoperoxidase analysis were subjected to deparanization

and dehydration by passing through a series of graded xylene and

alcohol, followed by a 5–min wash in distilled water. The slides

were then immersed in phosphate buffer solution (PBS, pH 7.2) for

5 min and subsequently treated with 3% H

for 10 min to inhibit

endogenous peroxidase activity. Following this, the slides were rinsed

in PBS for 5–10 min and incubated with a protein–blocking solution

compatible with all primary and secondary antibodies for 5 min to

TABLE II

Values obtained with the histopathological,

immunohistochemical, and immunouorescence staining

in liver tissues taken from average 65 days old rats

Histopathological

Parameters

Immunohistochemical

Parameters

Deg Inf Bh Fd

IHC/IF

Gld

IHC/IF

IgA IgG CD4 CD8

Wheat Group

1 +2 +2 +2 +2 +2 +2 +3 +2 +1 +2

2 +2 +2 +1 +1 +2 +2 +3 +2 +2 +2

3 +2 +1 0 0 +2 +2 +2 +2 +1 +2

4 +1 +1 0 0 +1 +2 +2 +2 +1 +2

5 +1 +1 0 0 +1 +2 +1 +1 +1 +1

6 +1 +1 0 0 +1 +2 +1 +1 +1 +1

7 0 0 0 0 +1 +1 0 0 0 0

8 0 0 0 0 0 0 0 0 0 0

Corn Group

1 +1 +1 0 0 +1 +1 +2 +2 +1 +1

2 +2 +2 +1 +1 +1 +1 +2 +2 +1 +1

3 +1 +1 0 0 +1 +1 +1 +1 +1 +1

4 +1 +1 0 0 +1 +1 +2 +1 +1 +2

5 +1 0 0 0 +1 0 0 0 0 0

6 0 0 0 0 0 0 0 0 0 0

7 0 0 0 0 0 0 0 0 0 0

8 0 0 0 0 0 0 0 0 0 0

Soybean Group

1 +1 +1 +1 +1 +1 +1 +1 +2 +1 +1

2 +1 +1 0 0 +1 +1 +2 +1 +1 +1

3 +1 0 0 0 +1 0 +1 +1 +1 +1

4 +1 0 0 0 +1 0 +1 +1 0 +1

5 +1 0 0 0 0 0 0 0 0 0

6 0 0 0 0 0 0 0 0 0 0

7 0 0 0 0 0 0 0 0 0 0

Deg: Degeneration, Inf: Inammation, Bh: Biliary hyperplasia, Fd: Fat drop, TG:

Transglutaminase, Gld: Gliadin, IgA: Immunoglobulin A, IgG: Immunoglobulin G

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prevent non–specic background staining. After incubation, excess

blocking solution was removed, and without further washing, the

slides were exposed to primary antibodies, including CD4 (Catalog

No: BS–0647–R), CD8 (Catalog No: BS–0648–R), IgA (Catalog No:

BS–0648–R10491–R), IgG (Catalog No: BS–0392–R), gliadin (Catalog

No: NB600–54713374–R), and transglutaminase 2/TGM2 (Catalog

No: NB600–547). Depending on the specific primary antibody,

incubation was conducted either for 1 hour at room temperature

or overnight at +4°C. The slides were then washed twice in PBS,

each time for 5min, and incubated with a biotinylated secondary

antibody at room temperature for 10–30 min. After another PBS wash,

the slides were treated with streptavidin–peroxidase for 10–30 min

and washed again with PBS. The sections were then treated with

DAB (3,3–diaminobenzidine) as a chromogen for 5–10 min. Finally,

background staining was performed using Mayer’s hematoxylin for 1–2

min, followed by rinsing with tap water and mounting with a water–

based adhesive [1, 11].

Immunouorescence examination

All sections mounted on poly–L–lysine–coated adhesive glass slides

for immunouorescence analysis underwent deparanization and

dehydration by passing through a series of graded xylene and alcohol

solutions, followed by a 5–min rinse in distilled water. The slides were

then immersed in phosphate buffer solution (PBS, pH 7.2) for 5 min

before being treated with 3% H

for 10 min to inhibit endogenous

peroxidase activity. Afterward, the slides were rinsed in PBS for

5–10 min and incubated with a protein–blocking solution for 5 min

to prevent non–specic background staining. At the conclusion of

the incubation, any excess blocking solution was removed from the

slides, which were then directly treated with the primary antibodies

gliadin (Catalog No: NB600–54713374–R) and transglutaminase 2/

TGM2 (Catalog No: NB600–547) without additional washing. Depending

on the specic primary antibody, incubation was carried out either

for 1 hour at room temperature or overnight at +4°C. The slides were

subsequently washed twice in PBS, each time for 5 min, and then

incubated with a secondary uorescein antibody (FITC) in a dark room

for 45 min. Finally, the sections were washed with PBS, covered with

a coverslip, and examined under a uorescence microscope (ZEISS

AXIO Scope A1) (ZEISS AXIO Scope A1, Germany), with images captured

from the necessary areas [12].

Biochemical examination

On the 45

and 165

d of the study, venous blood samples were

collected from eight animals per study group into 10 mL glass tubes

containing a coagulation accelerator. In the Biochemistry Department

Laboratory at Atatürk University, Faculty of Veterinary Medicine, the

blood samples were centrifuged at 3,000 G and +4°C for 5 min. The

extracted serum was stored at -82°C until analysis. Serum levels of

AST, ALT, LDH, and ALP were measured using an automatic analyzer

with commercial test kits (Cobas 6000 analyzer, Roche) [13].

Statistical analyses

The statistical analysis of the results from this study was conducted

using the Statistical Package for the Social Sciences (SPSS) software

[14]. One–way analysis of variance (ANOVA) was employed to analyze

serum levels of AST, ALT, LDH, and ALP, while Duncan’s test was used

to determine the signicance of differences between study groups.

Histopathological and immunohistochemical changes in the liver were

assessed using the Kruskal–Wallis test, a non–parametric method.

Data were presented as mean ± standard error of the mean (SEM).

Values of P<0.05 and P<0.01 were considered statistically signicant.

RESULTS AND DISCUSSIONS

Findings pertaining to the histopathological, immunohistochemical,

and immunofluorescence parameters of the hepatic tissue on

day 45 of the study (at 65 d of age) are presented in TABLE II, the

graphic representation of these ndings is shown in FIGS. 1 and 2,

and the results of the statistical analysis of the ndings are given in

TABLE III. Furthermore, ndings pertaining to the histopathological,

immunohistochemical, and immunouorescence parameters of the

hepatic tissue on day 165 of the study (at 185 d of age) are presented

in TABLE IV, the graphic representation of these ndings is shown in

FIGS. 3 and 4, and the results of the statistical analysis of the ndings

are given in TABLE V.

Wheat Corn Soybean

H&EIgAIgGCD4CD8

FIGURE 1. Liver tissue, 65–day–old rat, histopathological appearance, H&E, Bar: 70 µm. immunohistochemical staining results, IHC–P, Bar: 70 µm

Effects of experimental wheat and corn gluten on liver tissue in rats / Terim Kapakin et al. _________________________________________

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The histopathological ndings detected in the hepatic tissue on

d 45 and 165 of the study (degeneration, necrosis, inammation,

biliary hyperplasia, fat droplets) were more severe in Group Wheat,

compared to the other groups, yet this numerical difference was

statistically insignicant (P>0.05) (TABLES III and V). Images of the

study are given in FIGS. 1 and 3.

In view of the length of period, during which the animals were fed

on the trial rations supplemented with wheat gluten, corn gluten and

soybean meal, it was observed that the severity of the histopathological

ndings in the hepatic tissue samples of all groups had signicantly

increased over time (165 d of feeding), and the severity was greatest in the

group, which was given wheat gluten (P<0.05). Furthermore, the hepatic

histopathological ndings of the group that received dietary soybean

meal were more severe, compared to the group fed on corn gluten.

On d 45 of the study, the immunopositivity rates determined for

anti–transglutaminase, anti–gliadin, IgA, IgG, anti–CD4 and anti–CD8

TABLE IV

Values obtained with the histopathological,

immunohistochemical, and immunouorescence staining

in liver tissues taken from average 185 days old rats

Histopathological

Parameters

Immunohistochemical

Parameters

Deg Inf Bh Fd

IHC/IF

Gld

IHC/IF

IgA IgG CD4 CD8

Wheat Group

1 +3 +2 +2 +3 +3 +4 +4 +2 +2 +3

2 +3 +2 +2 +3 +3 +4 +4 +2 +2 +3

3 +2 +2 +2 +2 +3 +3 +3 +2 +2 +3

4 +2 +2 +1 +2 +3 +3 +3 +1 +2 +3

5 +2 +1 +1 +2 +2 +3 +3 +1 +1 +2

6 +2 +2 +1 +1 +2 +2 +2 +1 +1 +2

7 +1 +2 +1 +1 +1 +1 +2 +1 +1 +2

8 +1 +1 0 0 +1 +1 +1 +1 +1 +1

Corn Group

1 +2 +2 +2 +2 +2 +2 +3 +1 +2 +2

2 +2 +1 +2 +2 +2 +2 +3 +1 +2 +2

3 +2 +2 +2 +1 +1 +2 +3 +1 +1 +2

4 +1 +1 +1 0 +1 +1 +2 +1 +1 +1

5 +1 +1 +1 +1 0 +1 +1 +1 +1 +1

6 +1 +1 +1 0 0 +1 +1 0 0 +1

7 +1 0 0 0 0 0 +1 0 0 0

8 +1 0 0 0 0 0 0 0 0 0

Soybean Group

1 +2 +2 +2 +2 +2 +2 +2 +1 +1 +2

2 +2 +1 +2 +1 +2 +1 +2 +1 +1 +2

3 +2 +2 +2 +2 +1 +1 +1 +1 +1 +2

4 +2 +2 +1 +1 0 +1 +1 +1 +1 +2

5 +1 +1 +1 +1 0 0 +2 0 +1 +1

6 +1 +1 +1 0 0 0 +1 0 0 +1

7 +1 0 0 0 0 0 0 0 0 0

Deg: Degeneration, Inf: Inammation, Bh: Biliary hyperplasia, Fd: Fat drop, TG:

Transglutaminase, Gld: Gliadin, IgA: Immunoglobulin A, IgG: Immunoglobulin G

TABLE III

Statistical values of the histopathological, immunohistochemical,

and immunouorescence parameters of the liver tissue

samples taken from average 65 d old rats

Parameters

Groups

P–values

Wheat Corn Soybean

Histopathological

Deg

± SEM 1.13 ± 0.295 0.75 ± 0.250 0.83 ± 0.166

0.482

Median 1.00 1.00 1.00

Inf

± SEM 1.00 ± 0.267 0.63 ± 0.263 0.33 ± 0.210

0.156

Median 1.00 0.50 0.00

± SEM 0.38 ± 0.263 0.13 ± 0.125 0.17 ± 0.166

0.733

Median 0.00 0.00 0.00

± SEM 0.38 ± 0.263 0.13 ± 0.125 0.17 ± 0.166

0.733

Median 0.00 0.00 0.00

Immunohistochemical and Immunouorescence

IHC/IF

± SEM 1.25 ± 0.250 0.63 ± 0.183 0.57 ± 0.202

0.066

Median 1.00 1.00 1.00

Gld

IHC/IF

± SEM 1.63 ± 0.263

0.50 ± 0.189

0.29 ± 0.184

0.001

Median 2.00 0.50 0.00

IgA

± SEM 1.50 ± 0.422 0.88 ± 0.350 0.83 ± 0.307

0.351

Median 1.50 0.50 1.00

IgG

± SEM 1.25 ± 0.313 0.75 ± 0.313 0.83 ± 0.307

0.390

Median 1.50 0.50 1.00

CD4

± SEM 0.88 ± 0.226 0.50 ± 0.188 0.50 ± 0.223

0.314

Median 1.00 0.50 0.50

CD8

± SEM 1.25 ± 0.313 0.63 ± 0.263 0.67 ± 0.210

0.198

Median 1.50 0.50 1.00

The values are given as mean ± standard error of the mean (SEM), n=8,

a, b

: Means in

the same line with dierent superscripts dier signicantly (*:

P<0.05), (**: P<0.01).

Deg: Degeneration, Inf: Inammation, Bh: Biliary hyperplasia, Fd: Fat drop, TG:

Transglutaminase, Gld: Gliadin, IgA: Immunoglobulin A, IgG: Immunoglobulin G

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antibodies, by means of the immunohistochemical examination of

the hepatic tissue samples, were found to be higher in the group

fed on wheat gluten, compared to the other study groups, yet

this difference was except for gliadin statistically insignificant

(TABLEIII). It was ascertained that the immunopositivity rates for

anti–transglutaminase, anti–gliadin, IgA, IgG, anti–CD4 and anti–CD8

antibodies increased over time (highest on day 165 of feeding) in all

groups with the highest rates having been determined in the group

fed on wheat gluten (P<0.05) (TABLE V). The images obtained in this

study are presented in FIGS. 3 and 4.

On d 185 of the study, the immunopositivity inase, anti–gliadin,

IgA, IgG, anti–CD4, and anti–CD8 antibodies by means of the

immunouorescence examination of the hepatic tissue samples,

were found to be higher in the group fed on wheat gluten compared

to the other study groups (TABLE V). It was ascertained that the

immunopositivity rates for anti–transglutaminase, and anti–gliadin

antibodies increased over time (highest on d 165 of feeding) in all

groups with the highest rates having been determined in the group

fed on wheat gluten (P<0.05) (TABLE V). The images obtained in this

study are presented in FIGS. 3 and 4.

The serum levels of the hepatic enzymes investigated in the present

study are given in TABLE VI. It was determined that while the levels

of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH)

signicantly differed from each other at 65 d of age, the levels of

aspartate aminotransferase (AST) and LDH showed significant

differences from each other at 185 d of age (P<0.05).

Today, gluten–containing foods have become an essential

component of the human diet and individuals carrying the genes

HLA–DQ2 and DQ8 confront various health problems, which are related

to both the digestion of the structural proteins of gluten, and the

metabolism of these proteins [9]. The gliadin peptides, which are

generated upon the breakdown of gluten in the digestive system,

bind to human leukocyte antigen (HLA) molecules, and thereby,

induce the onset of clinical signs and immunological processes.

It is known that the structural peptides of gliadin induce cellular,

Wheat Corn Soybean

TGM2/IHCTGM/IFGliadin/IHCGliadin/IF

FIGURE 2. Liver tissue, 65–day–old rat, immunohistochemical and immunouorescence staining results, IHC–P, Bar:70 um, IF, Bar: 50 µm

Effects of experimental wheat and corn gluten on liver tissue in rats / Terim Kapakin et al. _________________________________________

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humoral and inammatory responses in tissues [15]. Given that its

structural peptides are resistant to both proteases and proteolysis in

the gastrointestinal tract of coeliac disease patients, gliadin cannot

be fully digested, and eventually triggers intestinal T cells [15]. The

emergence and advance of autoimmune diseases increases directly

proportional to the length of the period of exposure to gluten [16].

Reactions arising from gluten sensitivity are observed primarily

in the intestines, but have also been reported in other organs and

tissues [7, 17, 18]. In the present study, it was observed that glutens

led to histopathological lesions in the hepatic tissue. Examination of

hepatic tissue samples demonstrated that gluten exposure had led

to the degeneration and necrosis of hepatocytes, biliary hyperplasia,

and an increased number of fat droplets. These ndings are also

observed in the event of intoxication, infection and stress [19]. These

ndings suggest that, similar histopathological ndings may develop

in the hepatic tissue of animals fed on various protein sources (wheat

gluten, corn gluten, soybean meal).

TABLE V

Statistical values of the histopathological, immunohistochemical,

and immunouorescence parameters of the liver tissue

samples taken from average 185 days old rats

Parameters

Groups

P–values

Wheat Corn Soybean

Histopathological

Deg

± SEM 2.00 ± 0.27 1.375 ± 0.183 1.50 ± 0.188

0.168

Median 2.00 1.00 1.50

Inf

± SEM 1.75 ± 0.164 1.00 ± 0.267 1.125 ± 0.295

0.096

Median 2.00 1.00 1.00

± SEM 1.25 ± 0.25 1.125 ± 0.295 1.125 ± 0.295

0.947

Median 1.00 1.00 1.00

± SEM 1.75 ± 0.366 0.75 ± 0.313 0.875 ± 0.295

0.110

Median 2.00 0.50 1.00

Immunohistochemical and Immunouorescence

IHC/IF

± SEM 2.25 ± 0.313

0.75 ± 0.313

0.71 ± 0.360

0.066

Median 2.50 0.50 0.00

Gld

IHC/IF

± SEM 2.63 ± 0.420

1.13 ± 0.295

0.71 ± 0.286

0.001

Median 3.00 1.00 1.00

IgA

± SEM 2.75 ± 0.366

1.75 ± 0.41

1.125 ± 0.295

0.351

Median 3.00 1.50 1.00

IgG

± SEM 1.375 ± 0.183

0.63 ± 0.183

0.50 ± 0.189

0.390

Median 1.00 1.00 0.50

CD4

± SEM 1.50 ± 0.189

0.875 ± 0.295

0.625 ± 0.183

0.314

Median 1.50 1.00 1.00

CD8

± SEM 2.375 ± 0.263

1.375 ± 0.83

1.50 ± 0.189

0.198

Median 2.00 1.00 1.50

The values are given as mean ± standard error of the mean (SEM), n=8,

a, b

: Means in

the same line with dierent superscripts dier signicantly (*:

P<0.05), (**: P<0.01).

Deg: Degeneration, Inf: Inammation, Bh: Biliary hyperplasia, Fd: Fat drop, TG:

Transglutaminase, Gld: Gliadin, IgA: Immunoglobulin A, IgG: Immunoglobulin G

TABLE VI

Statistical values of the liver enzyme levels of the serum tissue samples

Parameters

Groups

P–values

Wheat Corn Soybean

65 days of age

AST 350.000 ± 35.972 392.000 ± 30.430 364.000 ± 30.100 0.655

ALT 541.000 ± 136.125

227.000 ± 23.791

211.000 ± 27.019

0.023

AST/ALT 0.65 1.73 1.73

ALP 155.600 ± 21.951 128.800 ± 29.778 184.400 ± 16.690 0.282

LDH 401.800 ± 32.346

516.600 ± 21.919

535.600 ± 12.258

0.004

185 days of age

AST 400.000 ± 13.928

516.000 ± 37.736

490.000 ± 23.749

0.025

ALT 179.000 ± 73.034 241.000 ± 59.414 213.000 ± 66.888 0.808

AST/ALT 2.24 2.14 2.30

ALP 133.400 ± 14.497 105.500 ± 13.093 166.800 ± 25.498 0.135

LDH 451.800 ± 19.059

554.800 ± 26.097

507.200 ± 10.283

0.010

The values are given as mean ± standard error of the mean (SEM). n=8,

a, b

: Means

in the same line with different superscripts differ significantly (

P<0.05, P<0.01).

AST: aspartate aminotransferase, ALT: alanine aminotransferase and ALP: alkaline

phosphatase, LDH: lactate dehydrogenase

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It was ascertained that, on days 45 and 165 of the study, the

differences between the increases in the histopathological ndings

of the hepatic tissue (degeneration, necrosis, inammation, biliary

hyperplasia and fat droplets) were statistically insignicant (TABLESIII

and V). On the other hand, on both d 45 and 165 of the study, values

pertaining to the severity of the histopathological ndings in the

hepatic tissue, including degeneration, necrosis, inammation,

biliary hyperplasia and fat droplets, were numerically higher in Group

Wheat, compared to the other study groups, and this suggests that

the hepatic tissue is more sensitive to wheat gluten. It is known that

the prolonged consumption of gluten–containing foods increases the

risk of gluten–related diseases in humans [16, 20].

Immunohistochemical examinations are essential to determining

histopathological tissue lesions, which develop in response to

gluten sensitivity. Immunohistochemical examinations enable the

determination of immunopositivity at cellular and tissue level, as

well as, the localization and distribution of specic cell components.

These examinations are based on the identication of specic cell or

tissue components by means of antigen–antibody reactions [21]. The

agreement of the results of these analyses with those of conventional

pathological and cytological examinations and other ndings is highly

important [22, 23].

Tissue anti–transglutaminase antibodies are produced by means of

a complicated mechanism. Glutens, found in the structure of cereal

proteins (and primarily gliadins), are perceived by tissues as antigens.

Thus, T cells are stimulated for the synthesis of antibodies against these

antigens. This results in increased levels of anti–transglutaminase

antibodies in tissues. A typical example to this is the increased levels

of anti–transglutaminase antibodies (tTG) in the hepatic tissue of

humans, who carry the genes HLA–DQ2 and DQ8 and consume wheat

gluten [9, 24, 25]. In the present study, although no marked increase

was detected in the hepatic tissue anti–transglutaminase antibody

levels of 65 days old rats fed on glutens, it was observed that sensitivity

reactions increased in the hepatic tissue over time and with prolonged

feeding on gluten (165d). Furthermore, the highest tissue levels of

anti–transglutaminase antibodies were determined in the group fed on

wheat gluten. The present study, which was carried out on healthy rats,

demonstrated that gluten sensitivity is not only observed in humans

carrying the genes HLA–DQ2 and DQ8, such that similar gluten–induced

reactions can be observed in the hepatic tissue of animals not carrying

these genes. In a study conducted on rats, it was reported that tissue

transglutaminase 2 levels increased in groups given wheat and corn

gluten, and immunopositivity for transglutaminase antibody was

observed in hepatocytes, inammatory cells, and epithelial cells of

the glands [1]. In another study, immunohistochemical examination

of liver tissues in celiac patients revealed that transaminase antibody

release was increased, particularly in endothelial cells and periportal

hepatocytes [9].

While it is reported that the level of anti–gliadin antibodies generally

increases in chronic diseases, 90% of untreated coeliac disease

patients also have increased tissue levels of anti–gliadin antibodies

[26]. The gliadin protein is presented to reactive CD4+T cells by

a T cell receptor, which triggers the production of cytokines that

cause tissue damage. CD4 and T cells increase the levels of T Helper

1 and T Helper 2, both of which are proinflammatory cytokines.

These cytokines, in return, bring about the generation of rstly B–

lymphocytes and then plasma cells. Plasma cells produce anti–gliadin

and anti–transglutaminase antibodies [26]. In the present study, the

level of immunopositivity for anti–gliadin antibodies in the hepatic

Wheat Corn Soybean

H&EIgAIgGCD4CD8

FIGURE 3. Liver tissue, 185–day–old rat, histopathological appearance, H&E, Bar: 70 µm. immunohistochemical staining results, IHC–P, Bar: 70 µm

Effects of experimental wheat and corn gluten on liver tissue in rats / Terim Kapakin et al. _________________________________________

8 of 11

tissue on d 45 and 165 of the study having observed to differ, points

out to the signicance of the length of the gluten exposure period

in the development of tissue sensitivity. The ndings of the present

study showed that the group displaying the highest sensitivity level

to anti–gliadin antibodies was Group Wheat. An important nding of

the present study was the increase that occurred in tissue sensitivity

with prolonged exposure to wheat gluten, even in healthy animals.

IgA is a primary component of many external secretions. Secretory

IgA molecules are particularly effective against microbial agents on

mucosal surfaces, and by preventing bacterial pathogens and their

toxins from adhering to mucosal epithelial cells, they neutralize these

agents. IgG is produced during the secondary immune response and

dominates the extracellular uid [27]. IgG antibodies act as opsonin

and activate the complement system via the classical pathway [28,

29, 30]. On d 45 of the study, the IgA and IgG levels of Group Wheat

were observed to be numerically higher. It was determined that the

statistical differences observed between the groups increased with

the prolongation of the period of exposure (period of feeding on

Wheat Corn Soybean

TGM2/IHCTGM/IFGliadin/IHCGliadin/IF

FIGURE 4. Liver tissue, 185–day–old rat, immunohistochemical and immunouorescence staining results, IHC–P, Bar:70 um, IF, Bar: 50 µm

_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34500

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gluten). Both IgA and IgG levels were observed to have signicantly

increased in the group fed on wheat gluten.

Hepatic enzyme activities are known to vary with several factors,

including among others disease, stress, pharmaceutical treatment,

environmental factors and nutrition. Damage to hepatic tissue, either

with a symptomatic or asymptomatic clinical course, leads to elevated

hepatic enzyme levels [31, 32, 33]

. Multiple studies have reported that

glutens cause damage to various tissues and organs, initially in the

intestines, in individuals carrying the genes HLA–DQ2 and DQ8 [1, 5].

The present study demonstrated the effect of gluten–containing feed

rations on liver enzyme activities. The investigation of the effects of

dietary wheat gluten, corn gluten and soybean meal on liver enzyme

activities in the study groups revealed no effect on AST and ALP

activities at 65 days of age, and interestingly, higher AST activity in

Group Wheat and higher ALP activity in Groups Wheat and Corn at

185 d of age. The ALT activity of Group Wheat determined to have

signicantly increased at 65 days of age and to have decreased at

185 d of age suggests the enzyme activity of the hepatic tissue to

Effects of experimental wheat and corn gluten on liver tissue in rats / Terim Kapakin et al. _________________________________________

10 of 11

have adapted to gluten exposure. In Group Wheat, LDH activity being

lower than that of Groups Corn and Soybean at 65 d of age, and being

signicantly higher than that of Group Soybean demonstrates the

impact of diet on liver enzyme activities. Furthermore, the AST/ALT

ratio having increased, and having exceeded a value of 2.00 in all

study groups shows that liver enzyme activities are not only affected

by diet, but also by age. In a study conducted on rats, it was reported

that in the group where corn gluten was used as a protein source,

serum AST and ALT levels decreased, while ALP levels and AST/ALT

ratio were similar to those in the control group [34].

CONCLUSIONS

It was ascertained that, compared to the groups that received

dietary corn gluten and soybean meal, the group fed on wheat gluten

presented with more severe adverse histopathological effects on the

hepatic tissue, including degeneration, necrosis and fat droplets.

Furthermore, it was determined that prolonged exposure increased

the adverse effects of dietary gluten on the immunohistochemical

parameters of the hepatic tissue. Wheat gluten was found to have

signicantly increased the immunopositivity levels for all of the

antibodies (anti–transglutaminase, anti–gliadin, IgA, IgG, anti–

CD4 and anti–CD8 antibodies) used in the immunohistochemical

examinations. It is considered that the ndings of the present study

will provide a reference for future studies on gluten metabolism and

autoimmune disorders.

Ethical approval

The experimental protocol of the study was approved by the Sivas

Cumhuriyet University Animal Experiments Local Ethics Committee’s

decision dated 2024 and numbered 08.

Conicts of interest

The authors have no declaration of competing interests.

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