Assessment of hemoglobin-dependent neurotoxicity: alpha-alpha crosslinked hemoglobin.

Adult human hemoglobin A0 (HbA0) has been shown to be neurotoxic, and we wish to report on similar studies conducted using a modified hemoglobin, which has been crosslinked between the alpha subunits (alpha-alpha Hb). Cortical cell cultures were prepared from fetal Swiss-Webster mice at 15-16 days gestation. Mature cultures (days in vitro, 12-16) were exposed to alpha-alpha Hb in a defined medium for 24-48 hours at 37 degrees C. Low micromolar amounts of alpha-alpha Hb were neurotoxic in a concentration-dependent fashion. This toxicity was attenuated by the antioxidants Trolox and U-74500A and by the iron chelator deferoxamine. The hemoglobin-binding protein, haptoglobin, also completely blocked alpha-alpha Hb-dependent neurotoxicity. The latter result was unexpected because complex formation between alpha-alpha Hb and haptoglobin was not detected using assays of haptoglobin fluorescence and hemoglobin peroxidase activity.