Lead and cadmium co-exposure modified PC12 viability and ER stress: study from a 3 × 3 factorial design

Although exposure to individual metal does exhibit its toxicity, combined exposures provide a more effective representation of the toxic effects of different heavy metal exposures on public health as well as ecosystems. Furthermore, there are few studies on composite exposure to low concentrations of heavy metals, which is more consistent with real-life exposure. The purpose of this study was to explore the neurotoxicity induced by combined exposure to low concentrations of Lead (Pb) and cadmium (Cd) and the potential interaction of their mixture in vitro.

PC12 cells were incubation with the corresponding concentration of cadmium chloride and/or lead acetate. Viability of PC12 cells was measured by CCK8 assay after 12, 24 and 48h incubation. Next, We measured the ROS, mitochondrial membrane potential (MMP) and apoptosis produced by different treated cells using ROS assay kit, JC-1 MMP assay kit and annexin V-FITC/propidium iodide (PI) apoptosis assay kit, respectively. Expression of proteins related to PI3K/AKT and endoplasmic reticulum (ER) stress in PC12 cells were tested by western blotting. Our study was the first to analyze the interaction between Pb and Cd using a 3 × 3 factorial design approach to observe neurotoxicity.

The results showed that the combined exposure of them was more cytotoxic than the single metal. The activation of PI3K/AKT signaling pathway and several parameters related to oxidative stress and ER stress were significantly altered in combined exposure to low concentrations of Pb and Cd compared with the Pb or Cd. Regarding apoptosis and ER stress, a synergistic interaction between Pb and Cd was evident. Moreover, evoked ER stress as a mechanism involved in the apoptosis of PC12 cells by the combined exposure to Pb and Cd.

The present study provides a theoretical basis used for the toxicological assessment of metal mixtures induced neurotoxicity of concern in terms of public health, and more effective control measures should be taken for the environmental pollution caused by various mixed heavy metals discharged from industry and agriculture.