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. 2019 Feb 20;8(3):373–380. doi: 10.1039/c8tx00340h

The effects of lead on the development of somites in chick embryos (Gallus gallus domesticus) under in vitro conditions: a histological study

Zahra Amini a, Naser Mahdavi-Shahri a,b, Roya Lari a,b,✉, Fatemeh Behnam Rassouli a
PMCID: PMC6505386  PMID: 31160971

graphic file with name c8tx00340h-ga.jpgLead (Pb) is one of the most abundant toxic metals in the environment that can cause a variety of harmful effects.

Abstract

Lead (Pb) is one of the most abundant toxic metals in the environment that can cause a variety of harmful effects. During embryonic development of vertebrates, somites are temporary organs that give rise to skeletal muscle, cartilage, tendon, endothelial cells, and dermis. In this study, we investigated the effects of lead on the development of somites and their derivatives in chick embryos under in vitro conditions. For this propose, fertilized eggs of Gallus gallus domesticus were incubated until they reached the stage of 15–20 somites. The somites and notochord were isolated and treated with different concentrations of lead acetate (500, 1000, 2000, and 4000 ng ml–1) for 72 h. Our results indicated that high concentrations of lead reduced the nucleus diameter, reduced the synthesis of collagen, inhibited the formation of the cartilage matrix in somite cells, and disturbed the formation and order of myotubes. In conclusion, the results of the current study for the first time indicated the disturbing effects of lead on the development of somites in the chick embryo. Our results revealed that lead disturbed the development of somites in the chick embryo, which suggested that at high concentrations it can cause a serious mortal danger to life.

Introduction

Lead (Pb) is one of the most abundant toxic metals in the environment that can cause a variety of harmful effects on both humans and animals. Lead is recognized as a sustainable element and a global danger for the environment.1 Humans may be exposed to lead through inhalation of polluted air, ingestion of food, and/or application of contaminated products such as cosmetics and lead-based paints.1,2 Even at low concentrations, lead is highly toxic, and it mainly accumulates in the liver,3 bones,4 brain,5 kidneys6 and muscles.7 It induces different effects, such as oxidative stress,7 carcinogenesis,8 disruption of calcium homeostasis, degeneration and neuronal dysfunction.9 Lead is also recognized as a genotoxic material due to its effects on chromosome stability, and they finally result in cancer.10 The half-life of lead varies in different organs, that is, it is 35 days in the blood, about two years in the brain, and can be decades in the bone.11 Lead can be transmitted to embryos from the third month of pregnancy,12 and analytical methods demonstrated that lead distribution in embryonic organs is similar to that in adult organs.13

A chick (Gallus gallus domesticus) embryo is one of the most widely used animal models with considerable similarity to mammalian embryos. Chick embryos can be easily removed from the yellow surface, and cultivated in vitro for further manipulation.14 During embryogenesis of the vertebrate, the segmental pattern is created when the somites are in a pattern produced from the paraxial mesoderm. Somites are temporary organs consisting of precursor populations of cells that give rise to important body structures.15 Somites appear as paired on both sides of the neural tube and notochord of the embryo. Tissues that surround somites play important roles in the formation, proliferation, differentiation, and survival of somitic cells.16 Following the interactions of somitic cells with surrounding tissues, the somitic cells are differentiated; their abdominal-interior parts generate sclerotomal cells, which eventually transform into cartilage and bone, while dorsal-lateral parts produce dermomyotome, which forms muscle and skin.17 In the chick embryo, the first pair of somites appears at 20 h. The rate of somitogenesis is a characteristic of species, as in Gallus gallus domesticus embryo, 52 pairs of somites form from day one to five, with a pair of somites forming every 90 minutes at 37 °C.16 Inductive signals from the notochord have been shown to be required for patterning of the somites and its future development.18

Several reports have indicated the negative effects of lead exposure on nervous system development during embryogenesis.19,20 It has been shown that lead affects epigenetic marks and could interfere with brain development and function.20 It could also reduce geometric and densitometric parameters, as well as the total thickness of articular cartilage.21 Experiments carried out on mammalian models indicated that lead decreased bone and body mass in young male rats,22 and reduced the mechanical endurance, growth plate thickness, and trabecular histomorphometry.21 Since effects of lead on the development of somites have not been reported yet, the aim of the current study was to investigate changes induced by lead exposure in the development of somites and their derivatives in the chick embryo under in vitro conditions.

Materials and methods

Experimental design

Fertilized chicken (Gallus gallus domesticus) eggs were obtained from a local hatchery. They were incubated at 38 °C and 70% humidity. At the stage of 15–20 somites, chick embryos were separated from the yellow surface as described before.23 Briefly, chick eggs were kept at room temperature for 15 minutes to reduce yolk disintegration. Eggshells were then removed using the window technique and embryos were separated from the yolk. Somites and notochord were isolated under a stereomicroscope (Carl Zeiss, Stemi 2000 C, Germany), and the extra-embryonic membranes were removed using fine needles and washed 3 times with phosphate-buffered saline (Biowest, USA). The embryonic somites were cultured in the presence of the notochord. For this propose, somite rows and notochords were transferred to a tissue-culture plate and cultured with Dulbecco's modified Eagle's medium (Biowest, USA) which was supplemented with 15% fetal calf serum (Biowest, USA), 1% antibiotics (penicillin and streptomycin) and different concentrations of lead acetate (Merck, Germany) (0, 500, 1000, 2000, and 4000 ng ml–1) as has been used before.23,24 The cultures were incubated at 37 °C with 5% CO2 in air for 72 h, while their medium was exchanged every 24 h.25

Histological studies

The somites were washed several times with physiological saline solution (Biowest, USA) at 72 h. Then the samples were fixed in Bouin solution (Merck, Germany) for eight hours and embedded in paraffin (Biowest, USA), sectioned at a thickness of 5 μm and stained with hematoxylin–eosin (Sigma-Aldrich, USA) to investigate the somite cell morphology.26 Meanwhile, carmine–picroindigocarmine (Merck, Germany) and picrofushin (Merck, Germany) staining protocols were used to study the presence of collagen fibers and the formation of myotubes,27,28 and toluidine blue (Merck, Germany) and alcian blue (Merck, Germany) staining methods were applied to investigate proteoglycans (PGs)29 and glycosaminoglycan (GAGs) of cartilage,30 respectively.

Transmission electron microscopy (TEM)

Untreated somites and those treated with lead acetate (4000 ng ml–1) were fixed with 2.5% glutaraldehyde (TABB Laboratories, UK) for 24 h, followed by several washing steps in 0.1 M sodium cacodylate buffer (PH 7.4 TABB Laboratories, UK). Then, samples were treated with 1% osmium tetroxide (TABB Laboratories, UK) for 1 h, washed again in 0.1 M sodium cacodylate buffer, and dehydrated through a graded series of ethanol (Biowest, USA). Samples were then embedded in resin (TABB Laboratories, UK), polymerized and sectioned with an ultramicrotome. The samples were examined under TEM (Zeiss, LEO 912 AB, Germany).

Statistical analysis

To evaluate abnormalities in somitic cells, the diameter of the nucleus was measured using the Image J software. Statistical significance was assessed by one-way ANOVA and Tukey's tests using the GraphPad InStat software. Data were presented as mean ± SD, and p values less than 0.05 were considered significant for all comparisons.

Results

Morphological alteration of somitic cells after lead treatment

Results indicated that upon hematoxylin–eosin staining, the majority of cells showed a spherical morphology with low cytoplasm and intact nucleus in the control (Con) group (Fig. 1A) and samples treated with 500 ng ml–1 lead acetate (Fig. 1B). In cells treated with 1000 ng ml–1 lead acetate, no obvious change in the cell density was observed, while the spherical morphology of cells changed and some nucleus destruction was observed (Fig. 1C). As shown in Fig. 1D and E, upon treatment of somatic cells with 2000 and 4000 ng ml–1 lead acetate, morphological alterations and nucleus destruction were obvious, while they were prominent at the highest concentration.

Fig. 1. Somitic cells after 72 h treatment with different concentrations of lead acetate. Control cells are shown in (A) and cells treated with different concentrations of lead acetate in (B–E). Semi-thin sections of somitic cells of the Con group (F) and cells treated with 4000 ng ml–1 lead acetate (G). Changes in somites are shown with H & E (A–E) and toluidine blue (F and G) staining. Scale bar: 50 μm for A–G. Arrows represent the somite cells.

Fig. 1

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Toluidine blue staining of semi-thin sections demonstrated that in Con group the somite cells were located close to each other with the circular nucleus. The division of some cells was observed (Fig. 1F). On the other hand, after treatment of samples with the highest concentration of lead acetate (4000 ng ml–1), some apoptosis signs, such as the destructed nucleus and changed morphology, were observed and also the density of cells was reduced (Fig. 1G).

Electron microscopy was used to examine the morphology of untreated somite cells and cells treated with 4000 ng ml–1 lead acetate. Confirming previous results, in the Con group (Fig. 2A), somitic cells presented a normal, dense and spherical morphology with an intact nucleus, and as shown in the higher magnification image (Fig. 2B), nuclei and intracellular organelles of untreated somite cells were morphologically normal. On the other hand, not only the density and number of somite cells reduced after treatment with the highest concentration of lead acetate but also the regular morphology of cells disappeared and cells became detached (Fig. 2C). In the higher magnification image, shrunk nucleus and damaged membrane and intracellular organelles of treated cells were obvious, and signs of degeneration and disorder of cellular components were observed (Fig. 2D), all of which indicate the probability of apoptosis in somite cells.

Fig. 2. Morphological alterations of somitic cells under TEM. Control cells (A, B) and cells treated with 4000 ng ml–1 lead acetate (C, D). Scale bar: 8 μm for A and C and 2.5 μm for B and D. Arrows represent the somite cells.

Fig. 2

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Reducing the effects of lead acetate on the nucleus size of somite cells

Examining the nuclei of somitic cells upon treatment with 500 ng ml–1 lead acetate did not indicate considerable changes, while after administration of 1000 ng ml–1 or higher concentrations of lead acetate, the diameters of nuclei reduced significantly (P < 0.001) compared to those of the Con group and those of the group treated with 500 ng ml–1 of lead acetate. In the groups treated with 2000 ng mL–1 and 4000 ng mL–1 concentrations of lead acetate, the size of nuclei was significantly smaller than that of the 1000 ng mL–1 concentration group (P < 0.001). Moreover, the diameters of the nuclei at the concentration of 4000 ng mL–1 of lead acetate decreased remarkably compared with those of the group treated with the concentration of 2000 ng mL–1 (P < 0.001) (Fig. 3).

Fig. 3. Nucleus diameter in somitic cells upon treatment with different concentrations of lead acetate. As shown, 1000, 2000 and 4000 ng ml–1 lead acetate significantly changed the nucleus size in comparison with the Con group or between indicated groups (***P < 0.001).

Fig. 3

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Inhibitory effects of lead acetate on collagen fibers

The effects of lead acetate on collagen fibers were examined by picrofushin and carmine–picroindigocarmine staining. As presented in Fig. 4, in Con samples and cells treated with 500 ng ml–1 lead acetate pink collagen fibers were observed, while collagen formation was dramatically reduced after treatment with 1000 ng ml–1 and 2000 ng ml–1 lead acetate, and it was inhibited or delayed in cells treated with 4000 ng ml–1 lead acetate.

Fig. 4. Collagen detection in somitic tissues upon treatment with different concentrations of lead acetate. Picrofushin (A–E) and carmine–picroindigocarmine (F–J) staining of Con cells (A, F) and cells treated with lead acetate (ng ml–1). Arrows show collagen fibers. Scale bar: 200 μm.

Fig. 4

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Similarly, carmine–picroindigocarmine staining revealed green collagen fibers in Con cells and cells treated with 500 ng ml–1 lead acetate. In addition, 1000 ng ml–1 lead acetate reduced the number of collagen fibers, and this effect was more obvious when 2000 and 4000 ng ml–1 lead acetate was applied (Fig. 4).

Reducing the effects of lead acetate on the formation of cartilage GAGs and PGs

Alcian blue staining was used for identification of GAGs in somitic tissue (Fig. 5A–E). Histological studies indicated the formation of blue GAGs in Con samples and cells treated with 500 ng ml–1 lead acetate. However, the amount of GAGs was reduced by 1000 ng ml–1 lead acetate, and after treatment with 2000 ng ml–1 and 4000 ng ml–1 lead acetate, no signs of glycosaminoglycan formation was observed. Accordingly, a decrease of GAGs following lead acetate treatment occurred in a dosage dependent manner.

Fig. 5. Detection of cartilage GAGs and PGs in somitic tissues after treatment with different concentrations of lead acetate. Con cells (A, F) and cells treated with lead acetate (ng ml–1). Arrows show GAGs and PGs in the intercellular matrix. Scale bar: 200 μm (A–E) and 50 μm (F–J).

Fig. 5

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To detect PGs upon treatment of somite cells, toluidine blue staining was used. As shown in Fig. 5F–J, PGs were observed in Con cells and cells treated with 500 ng ml–1 lead acetate, while 1000 ng ml–1 lead acetate reduced proteoglycan formation and in higher concentrations, this effect was considerable.

Negative effects of lead acetate on myotube formation

Myofibril like cell differentiation was examined by picrofushin staining. In the Con group, linear myofibril like cells were clearly observed (Fig. 6A), which indicated the differentiation of mesenchymal cells into myoblast cells. As shown in Fig. 6B, a number of these cells were multi-nuclear with a long morphology and linear position. When the cells were treated with a high concentration of lead acetate (4000 ng ml–1), the linear form of myofibrils was obviously reduced (Fig. 6C), and abnormal cells presented signs of nucleus destruction and probably apoptosis (Fig. 6D).

Fig. 6. Picrofushin staining of somitic cells for myogenesis detection. Con cells, (A, B) and cells treated with 4000 ng ml–1 lead acetate (C, D). Arrows show the myofibril pattern within the somite (B) and non-linear wavy myofibrils (D). Scale bar: 200 μm (A and C) and 50 μm (B and D).

Fig. 6

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Discussion

During embryonic development of vertebrates, somitic cells give rise to a wide range of tissues including skeletal muscle, cartilage, tendon, endothelial cells, and dermis.15 This study is the first attempt to report the effects of lead acetate on the developmental process of chick somites in vitro. The chick embryo has been widely used for developmental studies due to its similarity to the human embryo, in terms of morphology and development, and also its availability, short embryonic period and transparency for micro-surgery.31 Since notochord induction has been shown to be essential for somite differentiation18,32 and our primary experiments indicated that in the absence of notochord, somites did not grow and differentiate in vitro (data not shown), we used a co-culture of somites with notochord. The findings of the current study indicated that chick somites could be considered as suitable models to study the effects of pollutants on development under in vitro conditions.

Little research has been done to deal with the effects of lead on the embryonic stages of development. In humans, the effects of lead on embryo development are not clear, but a higher pregnancy failure in association with air pollution has been reported.33 However, in rats and mice lead has been shown to play a key role in the failure of embryonic development.34,35 Lead has also been shown to affect the methylation of DNA and neuronal differentiation of human embryonic stem cells.36 In the current study, investigating in vitro effects of lead acetate on the development of somites in the chick embryo indicated that it caused failure of somite cell development, including the synthesis of collagen, chondrogenesis, and myogenesis. Our data suggested the possibility of the lead effect on pregnancy failure in polluted cities.

In the current study, it has been demonstrated that exposure to high concentrations of lead acetate for 72 h induced morphological changes and nucleus destruction of somite cells. Previously, lead-induced apoptosis has been reported in different cells and tissues, for instance, in alveolar macrophage and also in human fibroblast cells.37,38 It has been suggested that the ability of lead to substitute for other bivalent elements such as calcium, is a possible triggering mechanism of various lead-induced fundamental biological processes such as intercellular signaling, cell adhesion, apoptosis, and ionic transportation.39,40 Electron microscopy also indicated the destruction of intracellular organelles upon administration of 4000 ng ml–1 lead acetate, and measuring the nucleus diameter in somitic cells revealed that lead acetate, at concentrations greater than 1000 ng ml–1, significantly reduced the nucleus size. Therefore, our data confirmed the cytotoxicity of lead as it also reduced the density and number of attached somitic cells, altered their regular morphology and damaged their membrane and intracellular organelles.

While most of the recent research studies have focused on the effects of lead in the developing peripheral and central nervous system, our results indicated that the influence of lead is far greater in developmental processes. Current results revealed that high concentrations of lead acetate delayed/inhibited the synthesis of collagen fibers. Since collagen is the main structural protein in many tissues such as bone and cartilage,41 and also the density and three-dimensional structure of collagen determine the mechanical properties of tissues and can act as a guide for cell migration,42 our results suggest impaired cellular migration that may result in the disruption of tissues and organs derived from somatic tissues during early stages of development. Previous studies have shown that lead exposure reduced collagen contents in the bone of rats43 and reduced the tendon size.44 In the current study, the reduction of collagen synthesis by lead could further interfere with the normal ossification and tendon development and can increase the risk of osteoporosis.

Previous studies indicated that although lead stimulates chondrogenesis, it reduces the maturity of chondrocytes and postpones the occurrence of endochondral ossification.45 Lead has been shown to reduce osteocyte and chondrocyte progenitors in bone marrow.46 Findings of the current study showed that lead acetate directly had inhibitory effects on the formation of the cartilage matrix in somatic cells, as confirmed by the reduced formation of GAGs and PGs in somites upon lead treatment. These results along with the decreased synthesis of collagen by lead could explain previous reports regarding inhibitory effects of lead on fracture healing.47

It has been demonstrated that lead exposure induced a curved body phenotype in zebrafish (Danio rerio) with concomitant changes in the somite length and abnormal muscle staining. In addition, molecular analysis showed decreased expression of the notochord marker (nt1) in the back–front axis of the zebrafish body after lead treatment.19 Since notochord signals affect somites, and consequently chondrogenesis and osteogenesis during sclerotome formation,15 the disruption of the cartridge matrix by lead in our study might be due to its negative effects on notochord signaling, and thus, reduced chondrogenesis. However, this possibility needs to be investigated by further research.

Myoblasts (progenitors of muscle cells) develop from somatic cells and finally differentiate to multinucleated myotubes.48 Hence, defects in somite cells result in developmental abnormalities that affect the axial curvature of the body.49 There is limited information concerning the pathogenic effect of lead on skeletal muscles. In zebrafish, for example, lead contamination negatively affects the development process of the muscle myofibril, as in treated embryos, the pattern of linear myofibrils was changed, some gaps appeared and myofibrils lost their linear form and turned into a wavy and curved pattern.19 Our results indicated that a high concentration of lead acetate changed the regular and linear structures of myofibrils, and destructed the morphology of cells and nuclei. There is some evidence which shows that the lead levels were related to a smaller birth weight or shorter length at birth.50 At high concentrations, lead could be mortal but at lower concentrations, it may damage or delay the development of myoblasts.

Conclusion

In the current study, we have presented the effects of lead on the development of somites and their derivatives in chick embryos under in vitro conditions. Lead caused the failure of synthesis of collagen and chondrogenesis and myogenesis of somatic cells. It also seems to induce apoptosis in the cells of the somites. Lead also reduced the density and number of attached somitic cells, altered their regular morphology and damaged their membrane and intracellular organelles. Our results indicated that lead had diverse influence on the developmental processes of somites. Therefore, the results of the current paper may open future perspectives of lead research. However, the mechanism of action of lead in somite cells must be explored. Our findings also revealed that somites of chick embryos could be used as a suitable in vitro model to study the effects of heavy metal cytotoxicity and teratogenicity and assess the influence of environmental polluting elements.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors of this article acknowledge the research deputy and the financial support from the Ferdowsi University of Mashhad (Grant number: 40870).

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