https://doi.org/10.52973/rcfcv-e34350

Received: 13/11/2023 Accepted: 21/01/2024 Published: 25/02/2024

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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34350

ABSTRACT

The study aimed to examine the effects of Azoxymethane (AOM),

Diallyl Disulde (DADS), and corn oil on the humerus and femur in

rats. In the study, 40 male Wistar Albino rats, 12 weeks old, were

used. The animals were divided into four different groups (Control,

AOM, DADS and Corn oil). After the experimental period, all animals

were anesthetized and sacriced by cervical dislocation. Humerus

and femur from long bones resected from all animal groups. The

maximum length, proximal width, distal width, diaphysis diameter,

cortex thickness and cavum medullare diameter of the bones were

measured. After the morphometric measurements index 1, index 2,

robusticity index, and bone weight/bone length index were calculated

for the bones. No difference was observed in the morphometric

measurements and indices performed on the humerus. A statistical

difference in proximal width and robusticity index was detected

between the groups in the femur. A difference was observed between

the AOM group and the Control/corn oil groups in proximal width. A

difference was detected between the AOM group and the Control

group in the robustness index, and it was determined that AOM caused

a decrease in density and strength in bones. It is thought that the

ndings obtained in this study will contribute to the evaluation of the

effects of cancer research using AOM on bones in rats.

Key words: Azoxymethane; corn oil; diallyl disulfide; femur;

humerus; morphometry

RESUMEN

El estudio tuvo como objetivo examinar los efectos del azoximetano

(AOM), el disulfuro de dialilo (DADS) y el aceite de maíz en el húmero y

el fémur de ratas. En el estudio se utilizaron 40 ratas Wistar Albinas

macho, de 12 semanas de edad. Los animales se dividieron en cuatro

grupos diferentes (Control, AOM, DADS y Aceite de maíz). Después

del período experimental, todos los animales fueron anestesiados y

sacricados mediante dislocación cervical. Húmero y fémur huesos

largos resecados de todos los grupos de animales. Se midieron la

longitud máxima, el ancho proximal, el ancho distal, el diámetro de la

diásis, el espesor de la corteza y el diámetro del cavidad medular de

los huesos. Después de las mediciones morfométricas, se calcularon

el índice 1, el índice 2, el índice de robustez y el índice de peso óseo/

longitud ósea para los huesos. No se observaron diferencias en las

mediciones e índices morfométricos realizados en el húmero. Se

detectó una diferencia estadística en el ancho proximal y el índice

de robustez entre los grupos en el fémur. Se observó una diferencia

entre el grupo AOM y los grupos Control/aceite de maíz en el ancho

proximal. Se detectó diferencia entre el grupo AOM y el grupo Control

en el índice de robustez, y se determinó que la AOM provocó una

disminución en la densidad y fuerza en los huesos. Se cree que los

hallazgos obtenidos en este estudio contribuirán a la evaluación

de los efectos de la investigación del cáncer utilizando AOM en

huesos de ratas.

Palabras clave: Azoximetano; aceite de maíz; disulfuro de dialilo;

fémur; húmero; morfometria

Morphometric investigation of the effects of Azoxymethane, Diallyl

disulde and corn oil use on humerus and femur development in rats

Investigación morfométrica de los efectos del uso de azoximetano, disulfuro de

dialilo y aceite de maíz sobre el desarrollo del húmero y el fémur en ratas

Sedat Aydoğdu

Selcuk University, Faculty of Veterinary Medicine, Department of Anatomy. Konya, Türkiye.

Corresponding author: sedatay85@gmail.com

Influence of Azoxymethane, Diallyl Disulfide, and Corn Oil on Rat Humerus and Femur Growth / Aydoğdu _________________________

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INTRODUCTION

The organic substances that make up the bone form the elasticity

of the bone, and the inorganic substances form the hardness and

durability of the bone [1, 2]. As progressive aging, the amount of

organic substance in the bones decreases, and the amount of

inorganic substance increases. For this reason, bone fractures are

more common in older animals [1]. This development and alteration

in bones is frequently investigated through experimental studies in

rats (Rattus norvegicus) [3, 4, 5, 6, 7].

Azoxymethane (AOM) is frequently used to create cancer models

in experimental animals. It has been reported that cancer models

created with AOM are similar to cancer observed in humans [8]. It

is stated that AOM induction changes the metabolic balance of the

intestinal epithelial layer and produces hydrogen peroxidase [9].

It is reported that garlic, which is known to have cancer prevention

properties, reduces stomach, breast, colon, esophagus and ovarian

cancers when used regularly [10, 11]. It has been stated that garlic

and garlic extracts have anti–cancer properties in animal models

[12]. Diallyl disulde (DADS), which is light yellow in color and liquid

with a pungent garlic odor, is one of the oil–soluble organosulfur

compounds of garlic. DADS is non–polar and insoluble in water. Due

to this feature, it can be dissolved in non–polar solvents [13, 14].

Therefore, DADS is applied by dissolving it in corn oil. Garlic and its

components, in addition to being antioxidants, also have antibiotic,

antiviral, antifungal, antihistamine, and anti–parasitic properties

[15, 16, 17]. Corn oil is obtained from Zea mays corn grains from the

Graminae family. Corn oil contains high amounts of unsaturated, low

saturated fatty acids and high amounts of vitamin E [18, 19].

Morphometry is one of the most important methods used to

measure length, width and thickness of biological structures such

as bones [20, 21]. Differences are revealed with the measurements

and statistical analyzes obtained with this method [20]. Morphometric

measurements in bones are made directly with a digital caliper. In

recent years, morphometric measurements have also been made on

3D models created using devices such as three–dimensional laser

scanners and computerized tomography [22, 23].

Morphometric measurements made on the front and hind leg bones

of rats have been used in sexual dimorphism as in other animals [24,

25]. It is reported that audiogenic stress negatively affects long bone

development in rats and signicantly reduces bone length [26]. The

effect of Methenolone Enanthate (ME), one of the Anabolic–androgenic

Steroids (AAS), on femur development was examined in adolescent rats.

In the study, it was observed that ME application negatively affected

femur length and corpus femoris thickness in male rats. [27]. It has

been reported that humerus and femur lengths were shortened in rats

supplemented with boldenone, trenbolone and testosterone [3, 5, 7].

In addition to the length measurement obtained from the bones, it is

stated that the robusticity increases at the same rate as the robusticity

index obtained from the bones decreases [24, 25, 28]. The effects of

lindane and linuron on bone morphemetry, calcium mechanism and

kidneys were examined in rats. It has been stated that the use of high

doses of linuron causes a decrease in the density and strength of the

femur [29]. Bone mineral content (BMC) and bone mineral density (BMD)

were examined in rats by dual–energy X–ray absorptiometry (DXA)

(Norland. Fort Atkinson. Wisc. USA). In the study comparing DXA and

histomorphometry with two morphometric indices (bone robusticity

and bone weight/bone length), it was determined that morphometric

indices were closely related to BMC [30]. It has been observed that

smoke inhalation has a negative effect on the skeletal system during

the development period, and the strength of bones decreases in rats

exposed to smoke inhalation [31]. Similarly, it has been stated that

estrogen deciency from ovariectomy causes bony tissue changes in

the femur length. Accordingly, it has been determined that the density

and strength of the femur decreases [32, 33, 34, 35].

The objective of the study is to morphometrically examine the

effects of AOM, DADS and Corn oil on the humerus and femur, which

are long bones, in rats. It is known that there is limited information on

the effects of AOM (It is frequently used to create cancer models in

experimental studies), DADS (It is one of the oil–soluble organosulfur

compounds of garlic), and Corn oil on long bones in the rat.

MATERIALS AND METHODS

In the study, 40 male Wistar Albino rats, 12 weeks old, were used. During

the study, animals were accommodated in environmental conditions of

40% humidity and 20°C, 12 hours at night and 12 hours during the day.

The study was approved by the Ethics Committee of Selçuk University

Faculty of Veterinary Medicine (Approval number: 2023/110 ).

Experimental design

The animals were divided into 4 different groups. In the rst group,

the Control group (n=10) the feed and water needs of the animals were

supplied ad libitum during the experiment (18 week). AOM group (n=10)

was fed a standard rat diet and 15 mg·kg

-1

body weight AOM (Azoxtmehane

Sigma–Aldrich) was injected subcutaneously (SC) 2 times for two weeks

[36]. DADS group (n=10) was fed a standard rat diet and 50 mg·kg

-1

body

weight DADS was administered for the last 3 weeks [37]. Since DADS is

non–polar and insoluble in water, it was applied by dissolving it in corn

oil. The corn oil group (n=10) was fed a standard rat diet and 1 mg·kg

-1

body weight corn oil was administered for the last 3 weeks.

Morphometric measurement

All animals were fed ad libitum during the experimental process (18

weeks). After the experimental period, all animals were anaesthetised

with Ketamine (95 mg·kg

-1

, SC) + Xylazine (5 mg·kg

-1

, SC) and sacriced

by cervical dislocation. Left humerus and femur resected from all

groups of animals. Since the effect of different substances used on

bone development was investigated in the study, the left/right factor

in bones was not taken into account. The soft tissue on the humerus

and femur of the rats was removed (FIG. 1).

After removing the soft tissue from the bones, measurements were

made using a 0.01 mm accuracy digital calliper (FIG. 2). Morphometric

measurement points performed on the femur and humerus are

specied in TABLE I. Morphometric measurements were carried

out from the measurement points applied in previous studies [4, 5,

7, 38, 39, 40]. The points of the morphometric measurements of the

humerus specied in TABLE I are shown in FIGS. 3.

In measuring the diaphysis diameter of the humerus, the lower

border level of the tuberosity deltoidea was considered. In measuring

the cortex thickness of the humerus, the average of four different

measurements of compact bone tissue was taken. In measuring the

cavum medullare diameter of the humerus, the mediolateral diameter

was considered as stated in previous studies.

FIGURE 1. Rat humerus and femur bones. A: Control group, B: AOM group, C:

DADS group, D: Corn oil group

FIGURE 3. Maximum length of the humerus (1), Proximal width of the humerus

(2), Distal width of the humerus (3), Diaphysis diameter of the humerus (lower

border level of the tuberositas deltoidea) (4), Cortex thickness of the humerus

{(5A+5B+5C+5D)/4} (5), Cavum medullare diameter of humerus (6)

FIGURE 2. Morphometric measurement with digital calliper. A: Humerus, B: Femur

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TABLE I

Morphometric measurements of humerus and femur

Parameters

Weight of bone Diaphysis diameter

Maximum length Cortex thickness

Proximal width Cavum medullare diameter

Distal width

The points of the morphometric measurements of the femur

specied in TABLE I are shown in FIG. 4.

FIGURE 4. Maximum length of the femur (1), Proximal width of the femur (2),

Distal width of the femur (3), Diaphysis diameter of the femur (lower border level

of the trochanter tertius) (4), Cortex thickness of the femur {(5A+5B+5C+5D)/4}

(5), Cavum medullare diameter of femur (6)

Influence of Azoxymethane, Diallyl Disulfide, and Corn Oil on Rat Humerus and Femur Growth / Aydoğdu _________________________

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In measuring the diaphysis diameter of the femur, the lower border

level of the trochanter tertius was considered. The points mentioned

in the humerus were taken into account when measuring the cortex

thickness and cavum medullare diameter of the femur.

The following equations of index 1, index 2, robusticity index,

and bone weight/bone length index were calculated with the

morphometric measurements obtained from the bones [24, 28, 30,

40, 41, 42]. The lower the index of robusticity, the denser and stronger

the bone. The higher the index of robusticity, the less dense or more

porous the bone [24, 42]. The bone weight/bone length index is a

simpler index of bone density used by Seedeor [41].

Index

Diaphysisdiameter

Diaphysis diameter Cavummedullare diameter

1 100

Index

Maximumlength

Diaphysis diameter

2 100

Robusticity index

Weigth of bone

Maximunlength

/Bone weight bone lengthindex

Maximunlength

Weight of bone

The data were evaluated in the statistical package program IBM

SPSS Statistics Standard Concurrent User V 26 (IBM Corp., Armonk,

New York, USA). Descriptive statistics are given as mean ± standard

deviation values. The homogeneity of the variances was tested using

the Levene test. Normal distribution of the data of numerical variables

was evaluated with the Shapiro Wilk normality test. One–way analysis

of variance was used if the data were normally distributed in the

variables, and the Kruskal Wallis H Test was used if the data were not

normally distributed (It was done only on the proximal width of the

femur parameter). Tukey test was used as multiple comparison tests

when the result of analysis of variance was signicant. p<0.05 was

considered statistically signicant. “Nomina Anatomica Veterinaria”

was used for anatomical terms [43].

RESULTS AND DISCUSSION

Humerus demographic results of all groups obtained from

morphometric measurements (weight of bone, maximum length,

proximal width, distal width, diaphysis diameter, cortex thickness,

cavum medullare diameter, index 1, index 2, robusticity index and

bone weight/bone length index) are summarized in TABLE II.

TABLE II

Descriptive statistics of parameters measured from the humerus (Mean ± SD)

Parameters Statistics

Weight of bone 0.45 ± 0.03

Maximum length 31.41 ± 0.62

Proximal width 6.20 ± 0.19

Distal width 7.60 ± 0.23

Diaphysis diameter 2.95 ± 0.11

Cortex thickness 0.93 ± 0.07

Cavum medullare diameter 1.14 ± 0.18

Index 1 61.27 ± 5.97

Index 2 9.38 ± 0.34

Robusticity index 4.09 ± 0.07

Bone weight/bone length index 14.47 ± 0.91

It was observed that the humerus lengths remained short in

rats administered different doses of boldenone, trenbolone, and

testosterone supplementation. Similarly, the use of testosterone

supplementation in young swim–trained rats causes shortening in

extremity bones [3, 5, 7]. In the study using AOM, DADS, and corn oil,

no difference in the length of the humerus was observed. Similarly,

It was observed that the use of Tarantula cubensis alcoholic extract

(TCAE) did not affect the length of the humerus.

Intergroup comparison of morphometric measurement results and

indices obtained from the humerus was presented in TABLE III. No

difference was observed between the Control, AOM, DADS, and Corn

oil groups in the measurements applied to the humerus.

In the indices obtained from the humerus, the highest values for

index 1 and index 2 were observed in the corn oil group. The highest

value in the bone weight/bone length index was observed in the AOM

group. In the robusticity index, where the index value and robusticity

are inversely proportional, the smallest value was calculated in the

AOM and DADS groups.

Femur demographic results of all groups obtained from

morphometric measurements are summarized in TABLE IV.

It was observed that the femur lengths remained short in rats

administered different doses of boldenone [3]. In another study

examining the effect of trenbolone supplementation on the extremity

bones of running rats, it was observed that the femur lengths were

short in rats [5]. Similarly, it has been reported that the use of

testosterone supplementation in young swim–trained rats causes

shortening in extremity bones [7]. In the current study, while no

difference was observed in the lengths of the femur, a difference

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Intergroup comparison of morphometric measurement results and

indices obtained from the femur was presented in TABLE V.

There is a statistically signicant difference between the groups in

the variables proximal width and robusticity index (P<0.05). There is a

difference between the AOM group and the Control/corn oil groups in

the proximal width parameter. The mean of the AOM group is higher

than the Control and corn oil groups. There is a difference between

AOM and Control groups in the robusticity index. The mean of the

AOM group is higher than the mean of the Control group.

In the indices obtained from the femur, the highest values for index

1 was observed in the AOM group. The highest values for index 2 was

observed in the DADS group. The highest value in the bone weight/

bone length index was observed in the Control group. In the robusticity

index, where the index value and robusticity are inversely proportional.

There is a difference between AOM and Control groups. The highest

robusticity index was observed in the AOM group. The use of AOM

negatively affects bone density.

Robusticity index, which is an important value in determining bone

density and porosity, was calculated in healthy female and male Wistar

albino, Wild rats and Sprague–Dawley rats [25]. The robusticity index

using different strains was calculated slightly higher than the values

obtained in the current study. It is thought that this difference is due

to the age of wild rats (ages unknown) and all domesticated rats (6

months) used in the relevant study.

Indices are calculated using morphometric measurements made on

bones. The robusticity index is one of the most frequently used indices

in research on rat bones. The lower the index, the denser or more robust

the bone [24, 25, 28]. In a study investigating the effects of lindane and

linuron on rats, it was determined that both compounds did not affect

the weight and length of the femur. However, it has been stated that the

use of high doses of linuron causes a decrease in density and strength

in the femur [29]. Similarly, it has been determined that the use of AOM

causes a decrease in density and strength in the femur. Additionally, it

has been reported that smoke inhalation and estrogen deciency from

ovariectomy in rats negatively affect bone development [32, 33, 34, 35].

TABLE III

Comparison of humerus parameters and indices between groups

Groups

Test Statistics P

Parameters Control AOM DADS Corn oil

Weight of bone (g) 0.46 ± 0.04 0.46 ± 0.04 0.45 ± 0.02 0.45 ± 0.04 0.390 0.761

Maximum length (mm) 31.67 ± 0.66 31.37 ± 0.70 31.20 ± 0.39 31.42 ± 0.69 1.009 0.400

Proximal width (mm) 6.23 ± 0.27 6.15 ± 0.14 6.19 ± 0.11 6.24 ± 0.22 0.395 0.757

Distal width (mm) 7.49 ± 0.20 7.62 ± 0.24 7.73 ± 0.29 7.57 ± 0.16 1.947 0.139

Diaphysis diameter (mm) 2.94 ± 0.08 2.94 ± 0.17 2.93 ± 0.08 2.97 ± 0.07 0.226 0.878

Cortex thickness (mm) 0.92 ± 0.05 0.94 ± 0.08 0.91 ± 0.06 0.95 ± 0.07 0.563 0.643

Cavum medullare diameter (mm) 1.22 ± 0.11 1.15 ± 0.18 1.09 ± 0.23 1.10 ± 0.18 1.005 0.402

Index 1 58.68 ± 3.28 60.78 ± 5.86 62.72 ± 7.44 62.88 ± 6.40 1.100 0.362

Index 2 9.30 ± 0.28 9.38 ± 0.52 9.40 ± 0.27 9.46 ± 0.25 0.351 0.788

Robusticity index 4.10 ± 0.07 4.07 ± 0.08 4.07 ± 0.06 4.11 ± 0.08 0.955 0.424

Weight / length index 14.55 ± 0.98 14.66 ± 1.06 14.43 ± 0.68 14.23 ± 0.96 0.393 0.759

Numerical variables are given as mean ± standard deviation.

: One–way ANOVA, Row–based lettering was done. There is no dierence between the

same letters. In the index equations, the weight is taken as milligrams

TABLE IV

Descriptive statistics of parameters measured from the femur (Mean±SD)

Parameters Statistics

Weight of bone 1.02 ± 0.07

Maximum length 41.66 ± 0.91

Proximal width 9.20 ± 0.32

Distal width 7.38 ± 0.18

Diaphysis diameter 5.14 ± 0.18

Cortex thickness 1.00 ± 0.08

Cavum medullare diameter 3.07 ± 0.18

Index 1 40.26 ± 3.38

Index 2 12.35 ± 0.35

Robusticity index 4.14 ± 0.06

Bone weight/bone length index 24.49 ± 1.33

was detected in the proximal width of the femur. It was observed

that the proximal width of the femur increased in rats given AOM.

Audiogenic stress is known to affect osteogenesis in rats, and

bone length negatively decreases signicantly. It is reported that ME,

one of the Anabolic–Androgenic Steroids, negatively affects femur

development and negatively affects femur length. Similarly, it was

observed that the humerus and femur lengths decreased signicantly

in rats given Methenolone Enanthate Supplement (MES) along with

exercise [6, 26, 27]. In the study using AOM, DADS, and corn oil, no

difference in the length of the femur was observed. However, it has

been observed that AOM negatively affects bone robusticity.

In another study, the effect of TCAE, which is used as a homeopathic

medicine in etherine medicine, on bone development in rats was

examined [4]. In the study where it was used in young rats, no

difference was observed in the measurements made on the femur,

as in the current study.

Influence of Azoxymethane, Diallyl Disulfide, and Corn Oil on Rat Humerus and Femur Growth / Aydoğdu _________________________

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TABLE V

Comparison of femur parameters and indices between groups

Groups

Test Statistics P

Parameters Control AOM DADS Corn oil

Weight of bone (g) 1.04 ± 0.10 1.00 ± 0.08 1.03 ± 0.04 1.02 ± 0.06 0.472 0.703

Maximum length (mm) 41.64 ± 1.44 41.84 ± 0.71 41.60 ± 0.60 41.56 ± 0.79 0.169 0.917

Proximal width (mm) 9.04 ± 0.39

9.40 ± 0.06

9.23 ± 0.26

9.15 ± 0.37

10.685 0.014

€

Distal width (mm) 7.32 ± 0.27 7.41 ± 0.14 7.38 ± 0.18 7.41 ± 0.12 0.522 0.670

Diaphysis diameter (mm) 5.05 ± 0.22 5.18 ± 0.17 5.18 ± 0.13 5.17 ± 0.16 1.220 0.316

Cortex thickness (mm) 0.99 ± 0.06 1.00 ± 0.06 0.98 ± 0.10 1.04 ± 0.09 0.937 0.433

Cavum medullare diameter (mm) 3.06 ± 0.21 3.04 ± 0.15 3.05 ± 0.24 3.14 ± 0.12 0.564 0.642

Index 1 39.43 ± 3.51 41.20 ± 3.16 41.05 ± 4.78 39.35 ± 1.04 1.692 0.639

€

Index 2 12.14 ± 0.41 12.37 ± 0.37 12.45 ± 0.34 12.44 ± 0.21 1.805 0.164

Robusticity index 4.11 ± 0.04

4.19 ± 0.07

4.13 ± 0.06

3.175 0.036

Weight / length index 24.91 ± 1.46 23.91 ± 1.63 24.67 ± 0.93 24.47 ± 1.17 1.040 0.386

Numerical variables are given as mean ± standard deviation.

: One–way ANOVA,

€

: Kruskal–Wallis test, Row–based lettering was done. There is no

dierence between the same letters. In the index equations, the weight is taken as milligrams

CONCLUSION

In this study, the effects of AOM, DADS and corn oil on bone

development were investigated, and morphometric measurements

were performed on the long bones humerus and femur. A difference

in the proximal width of the femur was observed in morphometric

measurements. According to the indices obtained from the

measurement results, it was observed that the use of AOM reduced the

density and strength in the femur. No difference was observed in DADS

and corn oil. It is thought that the ndings obtained in this study will

contribute to considering the effects of cancer studies on bones in rats.

Data availability statement

The data that support the ndings of this study are available from

the corresponding author upon reasonable request.

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