Protective effect of metallothioneins against oxidative stress evaluated on wild type and MT-null cell lines by means of flow cytometry

It is generally accepted that metallothioneins (MTs) are devoted to the regulation of the metabolism of essential trace metals and to chelation of toxic metals. Nowadays, there is increasing evidence that MTs also act as free radical scavengers. We employed wild type mouse embryo fibroblast cell line, GKA1, and its MT-null variant, GKA2, in order to correlate the presence of MTs to the sensitivity of cells to reactive oxygen species (ROS), spontaneously generated by the aerobic cellular metabolism, or chemically induced by hydrogen peroxide. The absence of MTs in GKA2 cells was unambiguously correlated to higher sensitivity to ROS attack, as evaluated by detection and quantification of 8-oxo-2′-deoxyguanosine (8-oxo-G), the first product of oxidative attack to DNA, using Fluorescence-Activated Cell Sorter (FACS). When compared to MT-null cell line, the wild type cells (GKA1) were less sensitive to ROS attack. In GKA1 cells, MT biosynthesis is readily induced by Cd ²⁺ treatment, and such an induction caused a further decrease in sensitivity to ROS injury. On the contrary, the MT-null cells (GKA2) expressed no detectable metallothioneins either constitutively, or after heavy metal pretreatment. Indeed, in GKA2 cell line, pretreatment with Cd ²⁺ did not reduce but even enhanced the oxidative stress.