An in ovo investigation of the cellular effects of the heavy metals cadmium and chromium alone and in combination

Abstract

Many heavy metals are essential for biological functions; however some of these metals, especially at high concentrations, can have serious adverse effects on humans. The main sources of heavy metal exposure are through agriculture, transport, mining and related operations. South Africa has a thriving mining industry and is known for its rich mineral resources, but due to the incorrect method of disposal of the waste from these mines, substances, including heavy metals, get into the water and air supply, affecting the people living in close proximity to these mines. Exposure is through inhalation of contaminated air and consumption of contaminated food and water. The most vulnerable to heavy metals are the developing fetus, because of the high rate of cell division and differentiation. In the current study, two heavy metals cadmium (Cd) and chromium (Cr) were chosen based on the possibility of being exposed to them in South Africa. Thus, the aim of this study was to investigate the possible cellular effects of the heavy metals Cd and Cr alone and in combination, at different concentrations, on brain, liver and kidney tissue by using a chick embryo model. This model was successfully implemented over a 14 day period after which the embryos were terminated and the brains, livers and kidneys removed and processed for light- and transmission electron microscopy (with energy dispersive spectroscopy and electron energyloss spectroscopy). In addition, the effect of Cd and Cr alone and in combination on DNA structure and micronuclei formation was evaluated. The levels of the major antioxidant component, glutathione was determined in the brains of the chick embryos. At low concentration of Cd and Cr alone and in combination, a hormesis effect was observed in the survival rates and weights of the chick embryos, while at x1000 physiological dose (PD) Cr and Cd alone and in combination the effects were toxic. The majority of viable embryos did not have any macro-anatomy abnormalities. Morphological evaluation of the brain, liver and kidney samples revealed that Cd caused severe alterations at its highest concentration with minor alterations at the lower concentrations. Cr and the metal combination groups on the other hand, only caused minimal alterations throughout the concentration ranges evaluated. The presence of Cd and Cr alone and in combination in the liver tissue was confirmed with the electron energy-loss spectroscopy analysis that detected these metals in the nuclei, mitochondria and Golgi complexes of the hepatocytes. This might contribute to the ultrastructural changes observed in this organ. The genotoxicity testing on the red blood cells revealed no substantial differences, as only a few micronuclei were present. Although heavy metals cause DNA damage through an indirect mechanism of oxidative damage, the presence of Cd and Cr in the nucleus and mitochondria indicates that these metals may have a direct effect on DNA structure. With DNA agarose gel electrophoresis it was found that Cd and Cr alone and in combination caused DNA fragmentation. In the brain, GSH levels were normal; however changes may be the result of Cd and Cr causing the depletion of other antioxidant elements such as glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase. In conclusion, this study indicates that Cd and Cr alone and in combination are toxic to the chick embryo. Cd is more toxic than Cr, and both metals accumulate in the nuclei and mitochondria where they induce damage either through oxidative and/or other mechanisms associated directly with DNA damage.