An in vivo and ex vivo investigation of effects of the heavy metals mercury and cadmium alone and in combination on the cardiovascular system

Abstract

South Africans living in mining regions and/or using contaminated water, both for drinking and irrigation, are the most vulnerable to the toxic effects of heavy metals. The effect of acute and chronic metal toxicity is well described although the knowledge of the effects on the cardiovascular system (CVS) is limited. The World Health Organization (WHO) has established limits for metals in water which for cadmium (Cd) and mercury (Hg) is 0.003 mg/L and 0.006 mg/L respectively. The aim of this study, using a male Sprague-Dawley rat model, was to investigate the toxic effects of both metals on the CVS and blood haemostasis. Sprague-Dawley rats, 6 per group were daily via gavage exposed to dosages equivalent to the 1000x, the WHO limits of Cd and Hg alone and in combination (Cd+Hg) for 28 days. This concentration was selected to identify specific cellular targets of toxicity in the CVS. After 28 days exposure that rats were terminated, the levels of each metal, several blood parameters as well as the tissue and cellular structure of the heart and aorta were evaluated. Tissue structure was evaluated using specific stains for the evaluation of general tissue morphology and the distribution of collagen and elastin. The ultrastructural features of both tissues were evaluated using transmission electron microscopy, with specific focus on the myocardium and collagen distribution in the heart, and collagen and elastin deposition in the aorta. With scanning electron microscopy the effect on blood cell morphology and haemostasis was also determined. The effect of the concentrations of Cd and Hg found in rat blood was further evaluated using a human ex vivo blood model. In the rat model, blood levels of Cd and Hg were increased. Rat weight and organ/weight ratio of the liver, kidney and heart was unchanged. Blood levels of alanine transaminase aspartate transaminase, urea nitrogen, glucose, total protein and globulin were unchanged. Compared to the control AP levels were reduced for Hg and Cd+Hg. For Cd+Hg, creatinine and sodium levels were also reduced while urea nitrogen was increased indicating that Cd+Hg causes some renal damage. In the myocardium, exposure caused mitochondrial damage and myofibrillar necrosis, increased fibrosis and the formation of lipid vacuoles. In the aorta, Cd caused alterations in elastin, with interruptions of elastin lamellae due to displacement by collagen. Hg alone and in combination caused collagen deposition as well as vascular smooth muscle cell proliferation and migration into the tunica intima, resulting in nonatherosclerotic neointimal plaque formation observed mostly in the Cd+Hg exposed rats. Exposure to Hg and Cd alone and in combination altered rat blood hemostasis and all metals caused platelet activation. Cd caused an increase fibrin fibre thickness, and induced aggregation and the formation of flat, fused areas of fibrin. Hg caused a decrease in fibre formation and for the combination group, a dense fibrin fibre network with extensive matted deposits between the fibres, with no changes in fibrin thickness. In whole blood the interaction of erythrocytes and fibrin fibres caused changes to erythrocyte morphology and fibrin network structure. Hg enhanced the effect of Cd on coagulation, resulting in the formation of dense, yet structurally less stable thrombi. Exposure to Cd and Hg alone and in combination was found to create a premature aging cardiovascular and prothrombotic blood phenotype that will increase the risk for CVD and thrombosis. In the ex vivo human blood model, Cd and Hg alone and in combination caused eryptosis and alterations in coagulation and erythrocyte and fibrin fibre interactions. Platelet activation was present with a significant increase in fibrin fibre thickness and aggregation, as well as formation of dense matted deposits, increasing the thrombotic tendency. Exposure to Cd and Hg alone and in combination also caused changes in erythrocyte morphology. The degree of platelet activation and changes to the morphology of erythrocytes were similar to that found in the rat animal model, which indicates that the ex vivo blood model can provide important although indirect information on the effects of exposure on the blood vascular system in humans. This model can be used as a screening method for heavy metal toxicity prior to animal studies. In conclusion, the CVS and blood is vulnerable to the effects of heavy metals such as Cd and Hg alone and as part of mixtures. Of concern is that markers of tissue damage do not reflect the extent of damage to the CVS. This study identifies that populations that are exposed to these metals are at increased risk for development of CVD and altered blood haemostasis although findings must be confirmed in exposed populations.