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THE EFFECT OF ORDINARY BENZENEON THE OSSIFICATION
CENTERS IN THE LONG BONES OF MICE EMBRYOS
Fawzi S.AL- Asadi Majdi Faisal Majeed Haifa Ali Hussan
*Department of Anatomy and Histology ,College of Veterinary Medicin,University of
Basrah,Basrah,Iraq.
(Received 21October 2013 ,Accepted 10 December 2013)
Key words: Mice embryo,Ossification centers, Benzene
ABSTRACT
Present study was to detect the ordinary benzene on ossification in the long bones of the
mouse embryos Musmusculus L.conducted the current study, 56 mice adult (16 male& 40
female) the females were divided into four groups, first for the control of the three treatment
groups wasexposed to ordinary benzene concentration (4.5ppm, 9ppm, 45ppm) hours / day
for 45 days and then married with intact males and took 20-day embryos,the results refer that
the treatment groups showed significant decrease p< o.o5 revealedin the centers of
ossification compared with control group.
INTRODUCTION
Benzene which is a volatile, colorless and highly flammable liquid was first discovered in
1825 by Michael Faraday, who isolated it from a liquid condensed from compressed oil gas
(1).
Benzene is found in the air from emissions from burning coal and oil, gasoline service
stations, and motor vehicle exhaust. Acute ( short – term ) inhalation exposure of humans to
benzene may cause drowsiness , dizziness , headaches, as well as eye , skin , and respiratory
tract irritation , and , at high levels , unconsciousness. Chronic (long – term) inhalation
exposure has caused various disorders in the blood, including reduced numbers of red blood
cells and a plastic anemia. In chronic exposures, benzene metabolites are considered the
chronic exposures, benzene metabolites are considered, the toxic agents, not the parent
compound (2). The relative contribution of different benzene metabolic pathways may be
dose related, with more toxic agents produced by high affinity low capacity pathways (3).
Chronic benzene exposure can cause bone marrow stem cell depression, apparently through a
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cytotoxic effect on all lineages of haematopoietic progenitor cells, although there is some
evidence for a mechanism involving injury to marrow stromal cells. Bone marrow
macrophages have been shown to metabolize phenol to reactive compound that bind
irreversibly to protein and DNA (4).
Benzene crosses the placenta and is present in cord blood in concentrations equal to or
greater than maternal blood (5). Animal experiment exposing pregnant mice and rats to
inhaled benzene in general demonstrated increased fetal skeletal variants and reduced fetal
weight, but failed to demonstrate consistent convincing evidence of teratogenicity. Rats
exposed to 313ppm for 24 hours/day on days 9 to 14 of gestation demonstrated reduced fetal
weight and increased skeletal variants (6).
During skeletal development, the majority of the bones in the body are established by
the endochondral bone formation process which is initiated by mesenchymal cell
condensation and subsequent mesenchymal cell differentiation in to chondrocytes and
surrounding perichondrial cells. Primary ossification occurs with osteoblast-mediated bone
formation, which initially occurs on the calcified cartilage template. Chondrocyte maturation
and the endochondral bone development process is factors, including bone morphogenetic
proteins (BMPs), fibroblast growth factors(FGFs), parathyroid hormone-related
protein(PTHrP) (7).
(8) reported that the hormone androgen in the chicken combines with estrogen to
cause ossification as that of the hormones the thyroid and neighbor thyroid and calcitonin
connection with the composition of the bone, because the thyroid hormone is important in the
process of differentiation and maturation of cartilage by stimulating the production of material
interstitial and the process of ossification it plays a role in stimulating inflation cartilage cells.
MATERIALS AND METHODS
Experimental Animals:
Used (56) adultMusmusculus mice(16 male, 40 female), 12weeks old, and of 23-26
grams weight were group housed in well ventilated cagesand provided with adequate food and
water.The light was adjusted in 12 h light/ dark cycle.
Thefemale mice were divided into four groups (10 mice each) exposed groups to
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concentrations (45ppm,9ppm and 4.5ppm) of ordinary benzene for 1 hour /day for 45days set
LC50, 13700ppm , the treatment groups mated with male control.
Determine the age of the fetus through the development of males with females in the
evening and isolated morning the next day, then females examined by investigating the
presence of sperm in the vagina supply only by injecting a physiological solution 0.9 %
sodium chloride in the females vagina and then take the contents through a special absorbent
and examined the contents placing them on a glass slide and optical microscopy as the day
has shown sperm per day zero (9).
Pregnant females sacrificed on day 20 of pregnancy after drugged with chloroform at room
temperature using anesthesia fund made was quickly out of the uterus and placed in a petri
dish contains 10% formalin , open the uterus and punctured embryonic membranes were
directed embryos from the uterus.
Determination of the primary ossification center.(for 20 day mouse embryos )
Primary ossification center length for long bones were determined by using method (
10 ).
1- Opens the skin and removed the viscera carefully avoiding the damage in bony or
cartilaginous skeleton or mesenchyme tissue.
2- Fixed the embryos at 100% ethanol for 4 days without stirring and then three days
with stirring.
3- imprisoned in the solution of Alizarin red and Alcian blue stain for five days with
stirring washed by tap water and imprisoned in solution of glycerol 20% with sodium
hydroxide 1% in ratio 1: 1 for 16h in room temperature to removed tissue completely
and stores in 15 glycerol for examined and photographed.
Using Ocular Micrometer, where the measured lengths ossification centers of the long
bones in the mid-body bone.
RESULTS
Effect of benzene on the ossification centers in the long bones
Table (1) and figure (1) show ossification centers in the long bones after exposed
to ordinary benzene in mice embryos in both the treated and control groups, Group A records
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significant decrease (p< o.o5) with the highest mean valve among all group. Al so Group A
shows a significant decrease (p< o.o5) when compared with control group.
The results in table(1) and figure(1) show more a significant decrease (p< o.o5) in the
ossification centers of ( humerus, radius ) bones of Group A compared with Groups B and C.
in addition, a significant decrease (p< o.o5) revealed in the ossification centers ulina bone of
Group A compared with Groups B and C, while no significant deference was observed
between Groups B and C as in figure ( 3, 4, 5 ).
The results show a significant decrease (p< o.o5)in the ossification centers femur
bone Group A compared with Groups B and C.in addition, a significant decrease (p< o.o5)
revealed in the ossification centers (tibia, fibula) bones of Group A compared with Groups B
and C as in figure (7, 8, 9).
The results showed in the 45pmm Group A some distortion in the ossification centers of long
bones in mice embryos marked by the lack of symmetry in the ossification centers of the long
bones as in figure (9).In addition, the lack of symmetry in the background of existing centers
of ossification from the right and the left as shown in figure (10, 11).
Fig.1. Values of ossification centers in the long bones of the exposed to ordinary benzene
in mice embryos.
.
Significantly different (p< 0.05)
GA: 45ppmGB: 9ppmGC: 4.5ppm
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Table .1.Values of ossification centers in the long bones of the exposed to ordinary
benzene in mice embryos.
Long
bones
Groups
Humerus Radius Ulina Femur Tibia Fibula
Control air
A
6.55
±
0.45
a
A
6.55
±
0.41
A
A
6.5
±
0.35
a
B
6.7
±
0.35
a
A
6
±
0.27
A
A
6
±
0.14
a
G A
A
2.55
±
0.31
b
A
2.57
±
0.51
B
A
2.5
±
0.14
b
A
2.5
±
0.35
b
A
3
±
0.23
B
A
3
±
0.13
b
G B
A
3.85
±
0.88
c
A
3.5
±
0.32
c
B
3.5
±
0.19
c
C
3
±
0.65
b
C
2
±
0.37
C
C
2
±
0.21
b
G C
A
4.2
±
0.91
d
A
4.5
±
0.31
d
A
3.5
±
0.34
d
B
3.5
±
0.31
b
B
2
±
0.31
C
B
2
±
0.23
c
*Significant differences at (P <0.05). Duncan's multiple range tests.
*Small letters refer to significant differences vertically.
*large letters refer to significant differences between rows.
*S. E. stander error
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Fig .3.Delayed formation of ossification centers (Humerus, Radius&Ulina) bones exposed of
benzene 4.5ppm in mice embryos (Alizarin red stain 140X)
Fig .2.Normal formation of ossification centers in (Humerus, Radius&Ulina)
bones of mice embryos. (Alizarin red stain 140X
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Fig .4.Delayed formation of ossification centers in (Humerus, Radius&Ulina) bones exposed
of benzene 9ppm in mice embryos.(Alizarin red stain 140X)
Fig .5.Delayed formation of ossification centers in (Humerus, Radius, &Ulina) bones exposed
benzene of 45ppm in mice embryos.(Alizarin red stain 140X)
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Fig .6:Normalformation of ossification centers in (Femur, Tibia&Fibula)bones in mice
embryos.( Alizarin red stain 140X)
Fig .7:Delayed formation of ossification centers in (Femur, Tibia&Fibula)bones exposed
benzene of 4.5ppm in mice embryos.(Alizarin red stain 140X)
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Fig .8:Delayed formation of ossification centers in (Femur, Tibia&Fibula) bones exposed
benzene of 9ppm in mice embryos. ( Alizarin red stain 140X)
Fig .9:Delayed formation of ossification centers in (Femur, Tibia&Fibula)bones exposed of
benzene 45ppm in mice embryos.(Alizarin red stain 140X)
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Fig .9: Delayed formation of ossification centers in (Femur, Tibia
&Fibula)bones exposed of benzene 45ppm in mice
embryos.(Alizarin red stain 140X)
Figure.11: show lack of symmetry in the ossification centers of (Tibia,
Fibula) bones between the right and the left, exposed benzene
45ppm in mice embryos (Alizarin red stain 140x).
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Figure.12: show lack of symmetry in the ossification centers of (Femur, Tibia, &
Fibula) bones between the right and the left, exposed benzene 45ppm in mice embryos
(Alizarin red stain 140x)
DISCUSSION
Effect of benzene on the ossification centers in the long bones of mice embryos:
In the present study showed that the effect of ordinary benzene on the
ossification centers in the long bones (Humerus, Radius, Ulina, Femur, Tibia, and
Fibula)were decrease on the ossification centers, these effect may be caused by
embryotoxicity of benzene.
(11) suggested that several factors may be responsis for the embryotoxicity of benzene :- first,
benzene can pass the placenta barrier and affect the embryonal cells directly; second, phenol
(a major metabolite of benzene) was shown to inhibit DNA synthesis in bone marrow in vivo
can also pass the placental barrier; third, benzene can damage the maternal circulation and
cause a bone marrow depression, resulting in adverse nutritional conditions for the fetus.The
bone marrow is a complex matrix harboring stem cells of blood cells, and stromal cells, which
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provide growth factors necessary for the proliferation and differentiation of stem and
progenitor cells (12).
(7) reported that the embryotoxicity caused a decrease of growth factors and
transcription factors (BMPs, FGFs, IHH, PTHrP and 2(RunX2) responsible for the
ossification centers of embryos mice.
(13) reported that the skeleton of the mice of the type 57B1 that mice-free protein Conexin
leads to delayed ossification membrane and cartilage and therefore the delay in the centers of
ossification indicating that this protein has an abundance of the barriers between cells
osteoblasts bones as the decreasing leads to lack of proteins responsible bone deposition
Therefore, this protein is important in the process of ossification and bone function of
osteoblasts and bone characterized this protein-free easily break and lack of diploe space.
تأثیرالبنزین العادی على مراکز التعظم فی العظام الطویلة لأجنة الفئران
فوزی صدام محسن مجدی فیصل مجید ھیفاء علی حسین
*قرع التشریح والانسجة ،کلیة الطب البیطری ، جامعة البصرة ، البصره ،العراق
الخلاصة
درس تأثیر استنشاق البنزین العادی على مراکز التعظم فی العظام الطویلة لأجنة فئران
اجریت الدراسة الحالیة على ٥٦ فأر بالغ ( ١٦ ذکر ، ٤٠ اناث ) قسمت الاناث الى اربعة مجامیع Musmusculus L.
4.5 )ساعة/یوم لمدة 45 ppm , 9ppm , 45ppm) الاولى للسیطرة وثلاث مجامیع عرضت الى البنزین العادی بترکیز
یوم بعدھا زوجت مع ذکور سلیمھ وأخذت الأجنة بعمر 20 یوم فکانت النتائج تشیر إلى أن المادة أحدثت انخفاض معنوی
فی مراکز التعظم الاولیة وازداد الانخفاض بزیادة الترکیز مقارنة مع السیطرةوقد تناسب التأثیر طردیا مع زیادة p<0.05
الترکیز .
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