Heavy metal toxicity

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Chapter 5: Effects of Cd and Hg alone and in combination on the blood coagulation system of Sprague-Dawley rats

Introduction 

Cardiac muscle tissue and blood vessels have been identified as targets of Cd and Hg toxicity. Likewise, following absorption, the blood cells are also exposed to these metals and exposure can also adversely affect blood haemostasis. Strong reciprocate interactions between the blood and the wall of blood vessels occur, and these physiological effects include changes in blood pressure and NO levels. The interaction between vessel walls and erythrocytes during coagulation facilitates the migration of platelets to the site of injury and the binding of inflammatory mediators to cell surface receptors (Pretorius and Kell, 2014).
The coagulation system plays an important part in the recovery of injured blood vessels and to prevent excessive blood loss through the formation of a blood clot or thrombus. The coagulation system therefore needs to be carefully controlled to ensure that unnecessary clot formation does not lead to a blockage of a blood vessels (Smith, 2009) or cause tissue damage in organs, as seen in complications such as CVA (Zhang et al., 2015). The phases of the cell-based coagulation pathway contribute to the formation of the clot with platelets playing a crucial role in this process (Pérez-Gómeza and Bove, 2007; Smith, 2009; Van Rooy, 2015), that through the release of certain factors regulates the tautness of the fibrin fibres (Pérez-Gómeza and Bove, 2007; Van Rooy, 2015).
Several individual metals such as iron (Fe) and Zn have been shown to have a pro-coagulative effect (Sangani et al., 2010) and Fe has been shown to alter the structure of fibrin networks (Pretorius and Lipinski, 2013) which implies that metal pollutants, as found in the environment, may also have an adverse effect on haemostasis and cardiovascular health. Studies on fibrin and thrombus formation provide physiologically relevant information about the possible risk for thrombosis associated diseases and a correlation has been found between in vitro and clinical studies (Undas and Ariens, 2011; Weisel and Litinov, 2013).
In the current chapter, the effects of Cd and Hg alone and in combination on the blood cells and coagulation system of Sprague-Dawley rats were investigated.

Materials and Methods

Materials

SEM was used to study erythrocyte, platelet and fibrin fibre morphology along with thickness of fibrin fibres. Blood from each animal in each of the experimental groups was prepared for whole blood (WB) and platelet-rich plasma (PRP) in order to examine the morphology of erythrocytes and fibrin networks respectively as described by Van Rooy et al., 2015.

Methods

Blood was collected in citrate tubes as described in Section 3.2.2.4 and transported to the Unit for Microscopy and Microanalysis, University of Pretoria, where it was processed for SEM.

Scanning electron microscopy preparation of platelets, fibrin networks and erythrocytes

A volume of 10 µl of WB, with and without the addition of 5 µl human thrombin (20 U/ml; South African National Blood Service), was added to 10 mm round glass coverslips (Leica SA). This preparation was used to evaluate the erythrocyte morphology prior to thrombin addition and the interaction of fibrin fibres with erythrocytes on addition to thrombi, as a representation of a thrombus. Blood was then centrifuged for 10 minutes at 227xg to obtain PRP. A volume of 10 μl of the PRP, with and without the addition of 5 μl of human thrombin, was placed on a 10 mm round glass coverslip. This preparation was used to evaluate platelet and fibrin network morphology. The glass coverslips were allowed to dry for 10 minutes and placed in 24 well plates to which 0.075 M sodium potassium phosphate buffer, pH 7.4 (NaP) was added. The samples were washed for 20 minutes on a shaker to remove any blood proteins. This was followed by fixing with 2.5% GA/FA solution for 30 minutes and rinsing the samples three times in NaP for 3 minutes before secondary fixation in 1% osmium tetroxide (OsO₄) for 15 minutes. The samples were washed again three times as described above. The samples were then dehydrated in 30%, 50%, 70%, 90% and 3 times in 100% ethanol. The SEM sample preparation was completed by drying the samples in hexamethyldisilazane (HMDS), followed by mounting and coating with carbon and viewing using the Zeiss ULTRA Plus FEG-SEM and the ZEISS Crossbeam 540 FEG-SEM (Carl Zeiss Microscopy, Munich, Germany).
The thickness of the fibrin fibres was measured to determine any alterations to the normal major (thick) and minor (thin) fibre arrangement of the network (Van Rooy et al., 2015). Fifty fibres were randomly chosen and measured on the SEM micrographs at X80000 using ImageJ (Version 1.49, Java).

Statistical analysis

Statistical analysis on fibrin fibre thickness were performed on GraphPad Prism Version 6.01 using 1-way analysis of variance (ANOVA), and Tukey‘s multiple comparisons test, where a p-value of ≤0.05 was considered significant.

Results

 In this section the effects of exposure to Cd and Hg alone and in combination on the morphology of blood obtained from the in vivo rat model are provided and changes are discussed in comparison to the blood obtained from the controls of the rat model. Effects on platelets are presented in Figure 5.1 while effects on the fibrin morphology and thickness are presented in Figures 5.2 – 5.6.Changes to erythrocytes are presented in figures 5.7 – 5.10 and to the thrombus morphology in Figure 5.11.

Platelet activation

Figures 5.1a – h are representative of platelets prepared from PRP in the control and exposed groups. In the control group (Figure 5.1a and b) round/oval platelets with some pseudopodia (arrows) were visible. Pseudopodia, change in platelet shape and spreading indicates platelet activation, which is not expected in the control group. The presence of pseudopodia in the control sample here is due to contact activation. (Van Rooy, et al., 2015). Platelet activation can be seen  in all the metal exposed groups (arrows in Figures 5.1c – h), with an increase in activation; relevant to the number of pseudopodia and amount platelet spreading, seen in the Cd (Figure 5.1c and d) and Hg (Figure 5.1e and f) and Cd and Hg groups (Figure 5.1g and h). Platelet-platelet interactions identified as aggregations of two or more platelets is observed in the Hg (Figure 5.1e and f) and in the combination groups (Figure 5.1g and h). Spontaneous fibrin fibre formation (Figure 5.1g) is  also indicative of increased activation and consequently thrombotic potential of the exposed groups.

Fibrin network morphology

Evaluation of the fibrin network in the control group (Figure 5.2a) revealed a typical fibrin network structure, with individual fibres of varying thickness in some cases overlapping to form a meshwork. As expected the fibrin fibres of the control group consisted of both thick, major fibres and thin, minor fibres (Pretorius et al., 2011). Fibrin fibres in the Cd group (Figure 5.3 a-d) appeared denser as compared to the control and formed areas of fused fibres as indicated by the arrows. Fusion (Figure 5.3a and b) and coiling (Figure 5.3d) of fibres were also observed The Hg group revealed a sparse formation of fibres (Figure 5.4a – d), the sample from this group with the most fibrin network formation is shown in Figure 5.4c, where thick fibres (thick arrow) and thin fibres (thin arrow) are seen in a sparse network. The Cd+Hg combination group (Figure 5.5a – d) revealed denser and more aggregated fibres compared to the control and have areas where the fibres appear fused (Figure 5.5c, arrow) with the presence of DMDs. Knot like structures were present within the networks (Figure 5.5d). Evaluation of the fibrin fibre thickness revealed that Cd only had a statistically significant increase in fibre thickness as compared to the control and the other two experimental groups (p-value ≤ 0.05) (Figure 5.6).

Erythrocyte morphology

Following platelet and fibre morphology, the morphology of erythrocytes was also evaluated. Erythrocytes from the control group (Figure 5.7a and b) have a rounded, biconcave appearance, typical of normal erythrocytes. The plasma membrane of the cell is smooth.
In the heavy metal exposed groups varying erythrocyte shapes were observed. Early elyptocyte shape, with elyptocyte formation was often observed in the Cd group (Figure 5.8) and membrane damage is visible in figure 5.8d. More extensive membrane damage was observed in the Hg group (Figure 5.9d). Spontaneous fibrin fibre formation was also observed around the erythrocytes in the Hg group (Figure 5.9c). Folding/crinkling of the plasma membrane were observed in the metal combination group on early elyptocytes (Figure 5.10a). Echinocytes and stomatocytes with tear drop morphology (Figure 5.10c and d) and membrane extensions were also observed (Figure 5.10d).

Thrombus morphology – Erythrocyte and fibrin interactions

The morphology of WB exposed to thrombin was evaluated in order to demonstrate interactions between the fibrin networks and erythrocytes in a blood clot. This also provides an indication of the tautness of the clot that forms. In WB samples with added thrombin, it was apparent that the metal exposed groups (Figure 5.11) showed the most erythrocytes damage when compared to the control (Figure 5.11a and b). Elyptocytes and early eryptotic cells were observed in the Cd (Figure 5.11c and d) and Hg groups (Figure 5.11e and f) while the combination group contained eryptotic cells (Figure 5.11g and h). Interactions between fibrin fibres and erythrocytes revealed that, unlike the control where fibrin fibres are smoothly wrapped around erythrocytes as shown in Figure 5.11b, in the metal exposed groups, the tension of fibres on the cell membranes was evident. As the erythrocyte membranes are damaged as indicated by the change in cell morphology, the folding of the cells around the fibres may be due to the membrane weakness as a result of the damaging effects of the heavy metals. In some areas the fibrin fibres of the Cd (Figure 5.11c, arrow) and combination group (Figure 5.11h) appeared straight and taut.

Discussion

Disturbances in the coagulation system will increase the risk or exacerbate pre-existing CVD in population exposed to contaminated water and other sources of environmental heavy metal exposure. Interactions between platelets and fibrin networks are necessary in the coagulation cascade and therefore the alterations in the structure of platelets and fibres can be used as an indicator of changes in the coagulation system. Erythrocytes have already been established as a cellular model for indication of pathology in the circulatory system (Swanepoel and Pretorius, 2012; Pretorius et al., 2016). Fibrin and thrombus formation in vitro and in vivo can be used to provide physiologically relevant information on diseases associated with thrombus complications as in vitro studies have previously been shown to correlate with clinical data (Undas and Ariens, 2011; Weisel and Litinov, 2013).

Platelet activation

Alterations to the platelet and fibrin structure can cause changes to the coagulation system (Lang and Qadri, 2012; Pretorius, et al., 2016). Platelet changes consistent with activation, as described previously (Kuwahara et al., 2002, Van Rooy et al., 2015), were observed in this study, at varying degrees in the two metal groups alone and to a higher extent in the combination group. The degree of activation was evaluated based on the degree of pseudopodia formation as well as degree of platelet spreading, relative to the control. Low doses of heavy metals have been shown to disrupt normal structure and function of rat platelets (Kumar et al., 2001). Platelets are known to increase clot elasticity (Carr and Alving, 1995), therefore these changes in platelet shape and membrane characteristics affect the formed thrombus, and have also been linked to inflammation (Van Rooy et al., 2015).

Abstract
Declaration
Acknowledgements.
Publications in the field and conferences attended
Table of contents
List of figures
List of tables
List of abbreviations, symbols and chemical formulae
Chapter 1: Introduction
Chapter 2: Literature review
2.1 Heavy metal toxicity
2.2 Heavy metals exposure in the South African context
2.3 Cadmium in the environment
2.3.1  Cadmium absorption, distribution, metabolism and elimination
2.3.2  Biochemical and cellular effects of Cd
2.3.3 Clinical effects of Cd
2.4 Mercury in the environment
2.4.1 Mercury absorption, distribution, metabolism and elimination
2.4.2 Biochemical and cellular effects of Hg
2.4.3 Clinical effects of Hg
2.5 Combinational studies: Hg or Cd and other heavy metals
2.6 Cellular formation of reactive oxygen species/ reactive nitrogen species
2.6.1 Oxidant/antioxidant enzymes
2.6.2 Effect of ROS on lipid, protein and DNA
2.7 Oxidative damage and the cardiovascular system
2.7.1 Erythrocytes and platelets
2.7.2 Endothelium
2.7.3 Cardiac and smooth muscle
2.7.4 Fibroblasts and fibrosis
2.7.5 Modes of cell death in the CVS as a result of oxidative stress
2.8 Aim and objectives
2.8.1 Aim
2.8.2 Objectives
Chapter 3: Implementation of the Sprague-Dawley rat model
3.1 Introduction
3.2 Materials and Methods.
3.2.1 Materials.
3.2.2 Methods
3.3 Results.
3.4 Discussion
3.4.1 Relevance of Cd and Hg blood levels
3.4.2 Liver toxicity
3.4.3 Kidney toxicity
3.4.4 Cd and Hg effects on hepatic and renal injury markers
3.4.5 Effects of co-administration of the two metals
3.4.6 Cardiac specific markers – a review of relevance.
3.4.7 Relationship with renal and vascular systems
3.5 Conclusion
Chapter 4: Effects of Cd and Hg alone and in combination on the heart tissue and aorta of Sprague- Dawley rats
4.1 Introduction
4.2 Materials and Methods
4.2.1 Materials
4.2.2 Methods
4.3 Results
4.3.1 Structural effects of Cd and Hg alone and in combination of the morphology of the heart
4.3.2 Structural effects of Cd and Hg alone and in combination of the morphology of the aorta
4.4 Discussion.
4.4.1 Collagen in heart tissue
4.4.2 Vascular remodelling of the aorta
4.4.3 Clinical consequence of cardiac and aortal remodelling during premature ageing
4.5 Conclusion
Chapter 5:Effects of Cd and Hg alone and in combination on the blood coagulation system of Sprague- Dawley rats..
5.1 Introduction
5.2 Materials and Methods
5.3 Results
5.4 Discussion
5.5 Conclusion
Chapter 6: Effects of Cd and Hg alone and in combination on haemostasis in an ex vivo human blood model
6.1 Introduction
6.2 Materials and Methods
6.3 Results
6.4 Discussion
6.5 Conclusion
Chapter 7: Conclusion
7.1 Introduction
7.2 Key findings
7.3 Relevance of findings
7.4 Limitations and future recommendations
Chapter 8: References
APPENDIX A
Ethical clearance
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