Background and aims:
The requirement of selenium for life and its beneficial role in human health has been known for several decades. However, the cellular response to damage depends on the chemical form of selenium, for instance in terms of the involvement of p53. It appears that apoptosis provides a protective response in case of heavily damaged DNA. Studies with p53-deficient cells indicate prolonged survival in spite of DNA damage after treatment with sodium selenite. Cadmium is a widespread environmental pollutant that is present in food and tobacco smoke, and has a very long biological half-life (10-30 years). Cadmium has been shown to interfere with DNA repair processes. In the case of p53, cadmium alters the conformation of p53, disturbs DNA binding and reduces transcriptional activity. The consequences are increased susceptibility towards other DNA damaging agents and endogenous mutagens. This thesis examined the impact of cadmium chloride on the cellular response to sodium selenite and selenomethionine. Special attention was given to selenite-induced apoptosis, which has been shown to be dependent of p53.
All the experiments were performed in a human colon cancer cell line (HCT 116), either p53-proficient or p53-deficient. Cytotoxicity of cadmium, sodium selenite and selenomethionine was examined by determination of cell number and colony forming ability. Gene expression of Bax was determined by using real time RT-PCR. The activity of the effector caspases 3 and 7 was determined using a Glo® Caspase 3/7 Assay (Promega). Nuclear localization of apoptosis inducing factor (AIF) was examined; the visualization of the apoptosis inducing factor was determined by fluorescence microscopy.
Cadmium attenuated the cytotoxicity of high sodium selenite concentrations, but enhanced the cytotoxicity of selenomethionine. Both effects were strongly p53-dependent. Gene expression of Bax was induced in a time- and dose dependent matter when treated with selenite, but this effect was abolished by cadmium. No increased activity of the effector caspases 3 and 7 was found in any of the treatments, which indicates caspase-independent apoptosis. Incomplete AIF immunofluorescence assay indicated a decreased luminescence AIF signal at high sodium selenite concentration in the presence of cadmium, however with no quantified data.
In conclusion, our results suggest that cadmium may increase the resistance to sodium selenite-induced apoptosis in vitro, depending on the p53-status of the examined cell. Possible mechanism is inactivation of p53, inhibiting p53-dependent apoptosis induced by sodium selenite exposure, in spite of DNA damage. In the absence of a functioning tumor suppressor, as seen in the p53-deficient HCT116 cells and in p53-proficient HCT cells pretreated with cadmium and altered into a “mutant”-like form, cells could accumulate DNA damage. Cells will survive without reducing cell proliferation, causing further genomic instability and increased risk of critical mutations. Concerns have been brought up about possible toxicities from long-term intake of selenium supplementation. Individuals particularly at high risk of cadmium exposure (e.g. smokers, vegetarians, children, females) might be susceptible towards the impact of cadmium on p53 and selenite-induced apoptosis.