Bax regulates production of superoxide in both apoptotic and nonapoptotic neurons: role of caspases.

A Bax- and, apparently, mitochondria-dependent increase in superoxide (O(2)(·-)) and other reactive oxygen species (ROS) occurs in apoptotic superior cervical ganglion (SCG) and cerebellar granule (CG) neurons. Here we show that Bax also lies upstream of ROS produced in nonapoptotic neurons and present evidence that caspases partially mediate the pro-oxidant effect of Bax. We used the O(2)(·-)-sensitive dye MitoSOX to monitor O(2)(·-) in neurons expressing different levels of Bax and mitochondrial superoxide dismutase (SOD2). Basal and apoptotic O(2)(·-) levels in both SCG and CG neurons were reduced in SOD2 wild-type (WT) cells having lower Bax concentrations. Apoptotic and nonapoptotic neurons from Bax-WT/SOD2-null but not Bax-null/SOD2-null mice had increased O(2)(·-) levels. A caspase inhibitor inhibited O(2)(·-) in both apoptotic and nonapoptotic SCG neurons. O(2)(·-) production increased when WT, but not Bax-null, SCG neurons were permeabilized and treated with active caspase 3. There was no apoptosis and little increase in O(2)(·-) in SCG neurons from caspase 3-null mice exposed to an apoptotic stimulus. O(2)(·-) levels in nonapoptotic caspase 3-null SCG neurons were lower than in WT cells but not as low as in caspase inhibitor-treated cells. These data indicate that Bax lies upstream of most O(2)(·-) produced in neurons, that caspase 3 is required for increased O(2)(·-) production during neuronal apoptosis, that caspase 3 is partially involved in O(2)(·-) production in nonapoptotic neurons, and that other caspases may also be involved in Bax-dependent O(2)(·-) production in nonapoptotic cells.

Rapid activation of antioxidant defenses by nerve growth factor suppresses reactive oxygen species during neuronal apoptosis: evidence for a role in cytochrome c redistribution.

Depriving mouse sympathetic neurons of nerve growth factor (NGF) causes their apoptotic death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) begins in these cells soon after NGF withdrawal. We investigated the effects on these ROS of adding NGF to cultures of NGF-deprived neurons. ROS levels were monitored with the fluorescent, redox-sensitive dyes CM-H2DCFDA and MitoSOX Red. The intensity of the former dye increases when it is oxidized by H2O2 and free radicals downstream of H2O2. MitoSOX Red is relatively insensitive to oxidation by H2O2 but is sensitive to oxidation by superoxide (O2*-). Withdrawing NGF increased CM-H2DCFDA intensity, indicating elevated H2O2-associated ROS. Re-exposure of cells deprived of NGF to NGF resulted in rapid suppression of these ROS. Neurons deprived of NGF also had increased MitoSOX Red intensities. Readdition of NGF had no effect on MitoSOX Red fluorescence. The suppression of CM-H2DCFDA-detected ROS by NGF was caused by a rapid activation of glutathione redox cycling. The most likely explanation for these findings is that mitochondria increased O2*- production after NGF withdrawal. The O2*- was converted to H2O2 by dismutation, and the H2O2 was detoxified by accelerated glutathione redox cycling. Our previous work shows that H2O2 induces cytochrome c to be released from mitochondria in NGF-supported sympathetic neurons, whereas antioxidants that detoxify H2O2 block cytochrome c redistribution in NGF-deprived neurons. Readdition of NGF also immediately inhibits cytochrome c release. We present evidence that this inhibition is mediated by the rapid activation of glutathione redox cycling by NGF.

Seroprevalence of Toxoplasma gondii in swine from slaughterhouses in Lima, Peru, and Georgia, U.S.A.

Toxoplasma gondii is an important pathogen transmitted by food, with raw or undercooked meat as the main foodborne source of toxoplasmosis. In Peru, 2-4 million people have antibodies to T. gondii. It is believed that more than 60 million people in the United States are infected with T. gondii. In this study, the prevalence of T. gondii in pigs from Peru and the United States was determined by Western blot. The presence of IgG antibodies to T. gondii from serum samples was determined. Blood samples were collected from 137 pigs at a slaughterhouse in Lima, Peru, and 152 pigs at a slaughterhouse in Georgia. Of the serum samples collected from swine, 27.7% (n = 38) from Peru and 16.4% (n = 25) from the United States were positive for T. gondii. Swine represent a significant source of human infection with T. gondii in Peru and the United States.