Hemin is cytotoxic, and contributes to the brain damage that accompanies hemorrhagic stroke. In order to better understand the basis of hemin toxicity in astrocytes, the present study quantified hemin metabolism and compared it to the pattern of cell death. Heme oxygenase-1 (HO-1) expression was first evident after 2 h incubation with hemin, with maximal expression being observed by 24 h. Despite the induction of HO-1, it was found that the proportion of hemin metabolized by astrocytes remained fairly constant throughout the 24 h period, with 70-80% of intracellular hemin remaining intact. A period of cell loss began after 2 h exposure to hemin, which gradually increased in severity to reach a maximum by 24 h. This cell loss could not be attenuated by the iron chelator, 1,10-phenanthroline, or by several antioxidant compounds (Trolox, N-acetyl-L-cysteine and N-tert-butyl-α-phenylnitrone), indicating that the mechanism of hemin toxicity does not involve iron. While these results make it unlikely that hemin toxicity is due to interactions with endogenous H(2)O(2), hemin toxicity was increased in the presence of supraphysiological levels of H(2)O(2) and this increase was ameliorated by PHEN, indicating that the iron released from hemin can be toxic under some pathological conditions. However, when H(2)O(2) is present at physiological levels, the toxicity of hemin appears to be caused by other mechanisms that may involve bilirubin and carbon monoxide in this model system.