Evaluation of heavy metal toxicity in eukaryotes using a simple functional assay

Abstract

Although carcinogenesis caused by metals has been intensively investigated, the mechanisms of action, especially at the molecular level, are still unclear. This work aimed to investigate Cd²⁺, Cu²⁺, Ni²⁺, Cr³⁺, and Zn²⁺ mutagenicity and its relationship with oxidative stress. We have applied the Functional Assay for the Separation of Alleles in Yeast (FASAY) with only minor modifications to detect p53 defects caused by metals. In this method, human p53-coding gene (TP53) expressed in Saccharomyces cerevisiae activates transcription of the ADE2 reporter gene. Yeast cells, expressing p53, were exposed to increased concentrations of metals and, then, plated on media supplemented or not with adenine. Yeast colonies containing functional p53 grow independently of adenine supplementation and colonies containing nonfunctional p53 are dependent on this nutrient. Mutations in the TP53 are implicated in the pathogenesis of half of all human tumors. According to our results, Cd²⁺ was found to be the most toxic metal and produced the highest oxidative damage to lipids and proteins. At low concentrations (40 μM), this metal decreased viability and completely inhibited cell growth, while higher concentrations were necessary to produce the same toxic effect by Cu²⁺, Cr³⁺, and Ni²⁺. Zn²⁺ showed no significant toxicity. Cd²⁺ strongly induced damages and altered the function of p53, while Cu²⁺, followed by Cr³⁺, showed lower percentages of p53-mutant colonies. Our results point towards a relationship between the loss of functional p53 protein and oxidative stress, a mechanism that can be associated with tumor formation induced by heavy metals in mammalian cells. By this adaptation of FASAY developed by us it is possible to easily and rapidly detect mutations caused by metals or other stresses.