Ca2+-dependent and independent mitochondrial damage in HepG2 cells that overexpress CYP2E1

CYP2E1-dependent mitochondrial damage, in the presence or absence of extracellular calcium, was investigated. HepG2 cells expressing CYP2E1 (E47 cells) were preloaded with arachidonic acid (AA), washed, and incubated with iron–nitrilotriacetate 1:3 complex (Fe–NTA) in minimum essential medium (MEM) (1.8 mM Ca2+) or Ca2+-free MEM (SMEM). Toxicity in SMEM was CYP2E1-dependent, necrotic, and lipid peroxidation-dependent. Intracellular calcium did not significantly change during the incubation in SMEM. Mitochondrial damage preceded the loss of plasma membrane integrity and was significant at 12 h of incubation, in coincidence with the toxicity. E47 cells treated with AA + Fe in MEM also showed a decline of mitochondrial membrane potential (ΔΨm) that preceded the loss of plasma membrane integrity, but starting at earlier times, e.g., 3 h than in SMEM. The decline in ΔΨm and the toxicity in both MEM and SMEM were inhibited by α-tocopherol and cyclosporin A, while the calpain inhibitor calpeptin was only effective in MEM. In conclusion, oxidative damage to mitochondria and the permeability transition plays a role in the CYP2E1-dependent toxicity of Fe + AA in HepG2 cells, both in MEM and SMEM. Ca2+ mobilization and activation of calpain contributes to the more rapid onset of mitochondrial damage in MEM, while oxidative damage and lipid peroxidation are involved in the Ca2+-independent later onset of mitochondrial damage.