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Histological study of effect of semicarbazide on the epiphyseal plate of the tibia and the shaft of femur of juvenile male albino rats

Okasha, Ebtsam F.; Elbakary, Reda H.

The Egyptian Journal of Histology: December 2011 - Volume 34 - Issue 4 - p 818–828
doi: 10.1097/01.EHX.0000407661.12366.f0
Original articles

Introduction Semicarbazide (SEM) is an azodicarbonamide byproduct present in glass jar packaged foods especially babyfoods. As a relatively high consumption of these products by infants can result in higher exposure compared with other consumers.

Aim of this work The aim of this work was to study the effect of SEM on the epiphyseal plate of the tibia and the shaft of the femur of juvenile male albino rats using both light and electron microscopes as the target sites of SEM are mainly cartilage and bone.

Materials and methods This study was carried out on 30 juvenile male albino rats divided into a control group and a treated group. Animals of the treated group were administered 40 mg of SEM orally once daily for 30 days. At the end of the experiment, the shaft of the femur and the epiphyseal plate of the tibia were dissected, decalcified, and prepared for light and electron microscopic examination.

Results Sections obtained from treated rats showed severe osteochondral lesions in the form of multiple tears and fragmentation of the epiphyseal plate, the formation of many notches on both periosteal and endosteal surfaces, and multiple intralamellar resorption cavities associated with many multinucleated osteoclast were seen in the shaft of the femur. Both chondrocytes and osteocytes showed many degenerative changes in addition to a reduction in the collagen fibers of the matrix of both cartilage and bone.

Conclusion and recommendation The present study indicated that SEM administration during the juvenile period induced multiple osteochondral histological alterations that resulted in decreased bone mass and defects in the development of the epiphyseal plate, which represents a major risk factor for development during early adulthood; thus, it is better to avoid food products sold in glass jars, especially during juvenile period.

Histology Department, Faculty of Medicine, Tanta University, Tanta, Egypt

Correspondence to Ebtsam F. Okasha, Histology Department, Faculty of Medicine, Tanta University, Tanta, Egypt Tel: +20 010 892 5878; fax: +0402762341; e-mail: ebtsamokasha@gmail.com

Received May 11, 2011

Accepted August 3, 2011

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Introduction

Semicarbazide (SEM) is a chemical used to make the plastic gaskets that seal the jar lids of a range of products, including fruit juices, jams, honey, baby food, pickles, sauces, mayonnaise, mustard, and ketchup, to prevent leakage and microbiological contamination of the jar contents. Furthermore, it has also been detected in seaweed-derived products, which are widely used as food additives. SEM is a decomposition product of azodicarbonamide (which belongs to the hydrazine family of chemicals). The latter is used as a flour treatment agent to improve baking properties; this compound may be a source of contamination with SEM [1,2]. SEM is also one of the break-down products of nitrofurans, a group of widely used veterinary drugs, and has been detected regularly in foods and flour treatment additives [3]. SEM is a white crystalline powder highly soluble in water [4].

The discovery of trace levels of SEM in bottled foods (especially baby foods) led to assessment of the safety of this compound by regulatory agencies worldwide. On the basis of the levels reported in food, the health risk appears to be very small. However, as a relatively high consumption of products in glass jars by infants can result in higher exposure compared with other consumers, the presence of SEM in baby foods is considered particularly undesirable [5].

Bone is a multifunctional organ, protecting soft tissues, working as levers for skeletal muscle action, and serving as the body’s major store of calcium. In addition, it supports hematopoiesis and houses the brain and spinal cord [6]. The epiphyseal plate or the growth plate is a cartilaginous tissue located toward the ends of children’s long bones entrapped between the epiphysis and the metaphysis of bone. The growth plate is responsible for creating a calcified cartilaginous template for bone deposition for the longitudinal growth of long bones through a process called endochondral ossification, involving chondrocyte proliferation at the proliferative zone, maturation or hypertrophy, apoptosis, and mineralization at the hypertrophic zone. Because the growth plate is the most fragile structure in a child’s developing long bones, injury of this plate is common [7]. As bone mass is lost throughout adult life as part of the aging process, a major risk factor for the development of osteoporosis is the failure to achieve normal peak bone mass during early adulthood [8].

It has been suggested that growing animals are more susceptible to the toxicity of SEM hydrochloride (SEM-HCl) than adults [9]. In addition, SEM administration during the juvenile period exerts pleiotropic effects on different organs including bone, cartilage, testes, ovary, uterus, thyroid, thymus, spleen, and adrenals, which represent the main developing organs as regard to juvenile toxicity [10], whereas other studies have reported that the toxicological effects of subchronic exposure to SEM-HCl are mainly observed in bone and cartilage [11]. In terms of these experimental data, it will be of great value to evaluate the effects of SEM on the cartilage and bone during the juvenile period.

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Materials and methods

Fifteen juvenile male albino rats aged 28 days (juvenile rats are 7 weeks old [12]) and weighing 40–50 g were used in this study. All animals were kept in clean, properly ventilated cages under similar environmental conditions and fed the same laboratory diet. The animals were divided equally into two groups: control and experimental groups. Animals of the control group were held without any treatment throughout the entire experiment, whereas animals of the experimental group were administered SEM (Aldrich Chemical Company Inc.) orally by a gastric tube once daily at a dose of 40 mg/kg body weight [10] for 30 days.

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At the end of the experiment

The animals were anesthetized with diethyl ether and perfused with 2% buffered glutaraldehyde. The femur and tibia of all animals were removed and immersed in buffered glutaraldehyde. The bones were dissected free of soft tissue, washed with saline, and the upper ends of the femur and tibia were cut and immersed in glutaraldehyde as a fixative at 4ºC. All the specimens were then decalcified in a daily exchange of EDTA for 3 weeks.

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For light microscopic study

Specimens were processed for the preparation of paraffin sections and stained with H&E and MT [13].

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For electron microscopic study

The samples of the decalcified bones were fixed in 5% phosphate-buffered glutaraldehyde (pH 7.3) for 2 h at 4°C and post fixed in 1% phosphate-buffered osmium tetraoxid for 30 min. Then, they were dehydrated and embedded in epoxy resin. Ultrathin sections were cut using an LKB ultramicrotome (Leica ultracut-UCT-Germany) and contrasted with uranyl acetate and lead citrate [14] for examination with a transmission electron microscope (Joel TEM 100-SX, Japan), at the EM Unit, Faculty of Medicine, Tanta University.

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Results

Results of examination of the epiphyseal plate

Light microscopic results

Examination of H&E-stained sections of the epiphyseal plate of the upper end of the tibia of control animals showed a normal appearance of this plate, which comprised different zones with a normal arrangement including a resting zone, a proliferative zone, a mature or a hypertrophied cartilage zone, and a calcified zone (Fig. 1). Sections obtained from treated rats revealed many lesions, including degeneration of chondrocytes of the resting zone with multiple destroyed empty lacunae (Fig. 2), in addition to detection of multiple tears. Widening of the proliferative zone and fragmentation in the epiphyseal plate as well as overcrowding and disarrangement of the chondrocytes were also detected (Figs 3 and 4).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Figure 4

Figure 4

Examination of MT-stained sections of control animals showed a homogeneous blue-colored ground substance of the epiphyseal plate (Fig. 5), whereas sections of treated animals revealed reduced staining intensity of the matrix, which showed faint coloration compared with the control one (Fig. 6), with detection of collagen fibers around the fragmented plates (Fig. 7). In addition, blood vessels in between the chondrocytes in the epiphyseal plate were also detected (Fig. 8).

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

Figure 8

Figure 8

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Electron microscopic results

Chondroblasts of the control group were present near the surface and appeared as ovoid cells that displayed a large nucleus with a prominent nucleolus and the usual organelles of protein-secreting cells (Fig. 9), whereas chondrocytes appeared large with a pale cytoplasm, a few organelles, and surrounded by a matrix. The matrix surrounding the cells is known as the capsular or the territorial matrix, whereas that present in between the chondrocytes is known as an interterritorial matrix (Fig. 10). However, examination of ultrathin sections in the epiphyseal plate of the treated group revealed large vacuoles in the cytoplasm of some chondrocytes (Fig. 11), whereas others had a dark cytoplasm containing multiple dilated rough endoplasmic reticulum (rER) with some degenerated mitochondria, and their nuclei appeared irregular and peripherally located with peripheral margination and condensation of their chromatin (Fig. 12). Shrunken chondrocytes with thin remnants of cytoplasm surrounded by cavity were also detected (Fig. 13).

Figure 9

Figure 9

Figure 10

Figure 10

Figure 11

Figure 11

Figure 12

Figure 12

Figure 13

Figure 13

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Results of examination of the shaft of the femur

Light microscopic results

Examination of H&E-stained sections of the femur of the control group revealed that the shaft was formed of a plate of compact bone containing many osteons. Each osteon comprised a central Haversian canal surrounded by concentric lamellae of collagen fibers and bone matrix with osteocytes inside their lacunae. Basophilic cement lines separated the newly formed lamellae from the old ones. Bone was covered from outside by the periosteum formed of outer fibrous and inner cellular layers and internally lined by endosteum that had only a cellular layer (Fig. 14).

Figure 14

Figure 14

Examination of H&E sections from treated animals revealed different forms of lesions. The most prominent lesion involved the presence of multiple intralamellar cavities, some containing fatty bone marrow and others containing granulation tissue, in addition to the formation of many notches on both periosteal and endosteal surfaces (Figs 15 and 16). Many multinucleated osteoclast were observed close to these notches and cavities (Figs 17 and 18). The cement lines were irregular and the bone matrix appeared less acidophilic than that of the control group with focal erosion of the periosteum (Figs 18 and 19).

Figure 15

Figure 15

Figure 16

Figure 16

Figure 17

Figure 17

Figure 18

Figure 18

Figure 19

Figure 19

Examination of MT-stained sections of the control group showed a high content of collagen fibers (blue color) in the matrix forming lamellae in trabecular bone (Fig. 20), whereas that of treated rats showed a decrease in collagen fibers in the trabecular bone, which appeared widely separated with numerous adipocytes in the bone marrow in between (Fig. 21).

Figure 20

Figure 20

Figure 21

Figure 21

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Electron microscopic results

Examination of ultrathin sections in the shaft of the femur of the control group revealed that the shaft comprised bone cells and matrix. The osteoblasts were present on the surface and surrounded by a small amount of collagen fibrils. Each cell contained many ribosomes, multiple mitochondria, and an active nucleus with extended chromatic (Fig. 22). In contrast, osteocytes with a scanty cytoplasm with less cytoplasmic organelles and relatively large less active nuclei were surrounded by lacunae and had many cytoplasmic processes. They were surrounded by many collagen fibers arranged in lamellae. The fibers in each lamella were parallel to each other and perpendicular to that of the adjacent lamella (Figs 23 and 24).

Figure 22

Figure 22

Figure 23

Figure 23

Figure 24

Figure 24

Examination of ultrathin sections in the shaft of the femur of the treated group revealed many degenerative changes mainly in osteocytes, as the cytoplasms of some of them showed multiple dilated rER (Fig. 25) and many electron-dense mitochondria (Fig. 26). Others were shrunken and lost their cytoplasmic processes (Fig. 27) and had irregular nuclei with more condensed chromatin (Figs 27 and 28). In contrast, the matrix appeared more electron lucent with few collagen fibrils (Fig. 28). The osteoclasts were active with irregular boundaries and contained many nuclei as well as electron-dense bodies (Fig. 29).

Figure 25

Figure 25

Figure 26

Figure 26

Figure 27

Figure 27

Figure 28

Figure 28

Figure 29

Figure 29

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Discussion

The recent discovery of the widespread use of SEM in global food industries and in baby food as a result of packaging contamination has focused attention on the toxicity of this substance [15]. The target sites of SEM-HCl toxicity were mainly cartilage and bone. Furthermore, epiphyseal cartilage is the potential target of SEM lathyrogen action (lack of mineralization) that is established at all dose levels [10]. The lesions were much more diverse and serious in young animals, as young animals generally consume more food per body weight during the growing period than adults [11]; thus, the present study was carried out to study the effect of SEM on cartilage and bone of juvenile rats.

The results of the present study indicated that SEM induced severe osteochondral histological alterations. In the treated group, there was widening and disorganization of the epiphysial cartilage with disruption of the various zones. Some authors [16] attributed these changes to abnormal widening of the zone of maturing cartilage, which caused the general enlargement of the plate. They added that the main characteristics of SEM are abnormal widening of the hypertrophic zone, with failure to progress to calcification and ossification. However, reduction of the staining intensity of the cartilage matrix reflected decreased polymerization of the cartilage matrix or alteration in the matrix properties caused by inhibition of cross-linking reactions of collagen [10]. It has been hypothesized that fissures specifically occurred in the sites subjected to body weight loading such as the anterior area of the tibia, due to alteration in the matrix properties, which reduce the strength of cartilage, leading to its fragmentation and fissure formation. The presence of collagen fibers around the fragmented plates (detected as an acidophilic zone around the fragments of the epiphyseal plate in H&E sections and as blue-colored fibers in MT-stained sections) was attributed to the poor integration of the extracellular matrix of these ‘repair tissues’ with the matrix of the damaged cartilage; it was also found that, if the cartilage was injured after the period of active growth, the defects were usually filled with connective tissue or fibrous cartilage [17].

Vascular invasion of the cartilage is a characteristic phenomenon of endochondral ossification. Therefore, angiogenesis, which is the formation of a microvascular network, in the cartilage is believed to play a fundamental role in endochondral ossification and growth plate formation [18]. Until recently, the cartilage vascular canals of the epiphyseal growth cartilage have not been associated with any specific disease [19].

In the current study, ultrastructural findings of cartilage of treated rats revealed many degenerative changes in chondrocytes in the form of many cytoplasmic vacuoles, dilated rER, degenerated mitochondria, and irregular peripherally located nuclei, with more condensation of their chromatin. In addition, the matrix appeared electron lucent, with a few irregularly arranged collagen fibrils. The endoplasmic reticulum (ER) is a major storage organelle for calcium and a site of synthesis and folding of secretory proteins and cell membrane proteins. ER is sensitive to changes in the internal and external environment of the cell; both physiological and pathological conditions may disturb the function of the ER, resulting in ER stress, given the fact that the chondrocyte, the only resident cell found in cartilage, is responsible for the synthesis and turnover of abundant extracellular matrix and might be sensitive to ER stress. In addition, the proper function of the ER is not only essential to the quality and quantity of protein expression but also to overall cell behavior. Prolonged ER stress can result in growth arrest of chondrocyte, apoptosis, and impaired cartilage matrix expression [20].

In terms of the bone, experimental data showed that SEM induced different forms of lesions, mainly the formation of many notches on both periosteal and endocortical surfaces and multiple intralamellar cavities containing granulation tissue in addition to the presence of many osteoclasts close to and inside resorption cavities. This was consistent with the previous investigators [21], who reported that SEM acts as an osteolathyrogen agent (chemicals that inhibit cross-linking of developing connective tissue fibers, leading to a reduction in bone mass). This explained the increased activity and number of osteoclasts in addition to thin trabeculae bone with loss of their connectivity that was observed in this study.

The decrease in collagen fibers and the reduction of the bone matrix that was observed in H&E sections as less acidophilia and in MT-stained sections as less bluish coloration, which was proven by an electron microscope as an electron-lucent matrix with few collagen fibers, were in line with the fact that SEM impairs cross-linking reactions of collagen and elastin, which are essential for maturation of connective tissues, including bones and blood vessels [21]. Other studies [23,24] reported that SEM acts as a lysyl oxidase inhibitor; these enzymes are members of the amine oxidase family, which are involved in the maturation of elastic lamellae and collagen fibers, assembly of collagen molecules into fibrils, and are also required for the formation of cross links. This was confirmed by other researchers [22], who have reported a reduction in matrix owing to disruption in the maturation and/or deposition of the connective tissue fibers.

Furthermore, SEM appeared to act as an endocrine disrupter, showing multiple specific mechanisms of action; it showed an antiestrogenic activity as serum estrogen levels were reduced in treated females [25]. As the estrogen hormone plays an important role in endochondral bone formation, particularly during pubertal growth [26], in addition, estrogenic compounds may accelerate polymerization and facilitate calcium binding, thereby stabilizing the newly synthesized collagen [27]. SEM antiestrogenic activity resulted in decreased bone formation and increased bone resorption. The structure of the bone is maintained by the relationship between bone resorption and formation; thus, activated bone resorption may play an important role in the pathogenesis of bone loss [28].

The appearance of irregular cement lines and less acidophilic newly formed bone was attributed to an increase in the rate of bone resorption over that of bone formation, leading to irregularity of the cement lines that separate the newly formed bone from the old one [29]. In terms of excess fat cells in the bone marrow, some animal studies and MRI studies suggest that there may be a relationship between abnormal lipid metabolisms and osteonecrosis, and another study [30] found that in steroid-induced osteonecrosis, the size of bone marrow fat cells increases significantly at an early stage.

The ultrastructure results of the femur in the current study demonstrated that the most prominent changes in osteocytes were mainly degenerative in the form of shortening of cytoplasmic processes, multiple dilated rER, many electron-dense mitochondria, and very irregular nuclei with condensed chromatin; in addition, other cells were also shrunken. It has been reported that SEM induces DNA damage through the formation of hydrogen peroxide; furthermore, SEM-derived free radicals also participate in DNA damage. DNA damage induced by these reactive species may be linked to the degenerative changes observed in oseocytes [31].

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Conclusion

The results of the present study showed that oral administration of SEM during the juvenile period in male albino rats induced multiple osteochondral lesions, leading to failure to achieve normal peak bone mass and defects in development of the epiphyseal plate, which represents a major risk factor for development during early adulthood.

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Recommendation

In light of these findings, it is recommended to avoid food products sold in glass jars, especially baby food, and consumers should be advised to use fresh products. In addition to informing the leaders of industry to eliminate SEM from the metal twist caps used with glass jars and identify alternative types of gaskets sealing jars.

Table

Table

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Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

bone; cartilage; juvenile rat; semicarbazide

© 2011 The Egyptian Journal of Histology