Vitamin E succinate enhances steatotic liver energy status and prevents oxidative damage following ischemia/reperfusion.

We have previously shown that treatment of steatotic livers with vitamin E succinate decreases liver injury and increases survival after ischemia/reperfusion (I/R). It is now understood that compromised energy status is associated with increased injury following liver ischemia in the setting of hepatic steatosis at least partially as a result of increased reactive oxygen species (ROS) and induction of mitochondrial uncoupling protein-2 (UCP2). Given the association between ROS, mitochondrial function, and UCP2, it was our goal to determine whether the protective effects of vitamin E succinate were associated with decreased ROS injury, down-regulation of UCP2, or improvement of ATP levels following I/R. To test this, leptin deficient (ob/ob) mice with steatotic livers that had received other 50 IU of vitamin E succinate supplement per day or control chow for 7 days were subjected to total hepatic ischemia (15 minutes) followed by reperfusion. We measured liver expressions of ATP, glutathione (GSH), and UCP2 as well as mitochondrial DNA damage. Vitamin E treatment decreased hepatic UCP2 expression and increased ATP and GSH levels prior to I/R. These levels were maintained at 1 hour after I/R. At 24 hours, while hepatic UCP2 expression, ATP, and GSH levels were similar to those of mice not receiving vitamin E, mitochondrial DNA damage was blocked. These results revealed that vitamin E succinate decreased hepatic UCP2 expression, reduced oxidative stress, and improved mitochondrial function in mice with steatotic livers before and after I/R, identifying mechanisms of protection in this setting.

DNA damage in brain mitochondria caused by aging and MPTP treatment.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment leads to marked depletion of dopamine (DA) levels in the nigrostriatal pathway and dopaminergic neuronal degeneration in caudate-putamen and substantia nigra. MPTP is believed to inhibit complex I of the electron transport system leading to the generation of reactive oxygen species. We sought to test the hypotheses that MPTP treatment: (1) leads to dopamine depletion; (2) causes extensive mitochondrial DNA damage, and (3) that these effects would be age dependent. The levels of dopamine and its metabolites, DOPAC and HVA were analyzed by HPLC equipped with electrochemical detection. DNA damage was measured by quantitative PCR in both mitochondrial and nuclear (beta-polymerase) targets from the caudate-putamen, substantia nigra and cerebellum regions of control and MPTP-treated mice. The age groups studied were 22 days and 12 months. MPTP produced no significant effect on the levels of dopamine and its metabolites in young mice whereas in old, there was a significant decrease in this neurotransmitter system after MPTP administration. These 12-month-old mice, when compared to the young mice, showed a significant increase in mitochondrial DNA damage in the caudate-putamen and cerebellum. The latter region also displayed a significant increase in DNA damage in a nuclear gene. After treatment with MPTP, there was an age-dependent increase in DNA damage in mitochondria of the caudate-putamen while there was no significant DNA damage in the nuclear target. MPTP treatment led to damage in both mitochondrial and nuclear DNA of the substantia nigra, while there was no damage in either mitochondria or nucleus in cerebellum which was used as a negative control.

Neurons in the cerebral cortex are most susceptible to DNA-damage in aging rat brain.

Using nick translation type of incubation and terminal deoxynucleotidyl transferase catalyzed 3 -end labeling, single and double strand breaks in genomic DNA of permeabilised neurons from different regions of young, adult and the aged rat brain were assessed. A steady increase in both types of breaks is noted with advancement of age in all of the brain regions studied. However, the number of SSB encountered in the cerebral cortex was the maximum and was also markedly higher than that in other brain regions. When the neuronal cells were exposed to MNNG or Glutamate the damage was aggravated in all the regions and at all ages but the most severe SSB damage is present in the cerebral cortex of older animals. Both cerebral cortex and the hippocampus showed equally higher DSB in comparison with the other regions. It is concluded that with advancement of age, DNA-damage accumulates in neurons and the cerebral cortex is the most vulnerable region.

Accumulation of DNA damage in aging neurons occurs through a mechanism other than apoptosis.

Two biochemical strategies using nick translation-type of incubation and terminal tranferase-catalyzed reaction were used to assess single-(SSB) and double-strand (DSB) breaks in DNA of permeabilized neurons isolated from young, adult, and old rat cerebral cortex. Both SSBs and DSBs accumulate with age. On prior treatment of neuronal cells with 1 mM glutamate or 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), more extensive damage was seen at all ages, with the old neurons suffering maximal damage. When neuronal DNA was subjected to agarose electrophoresis, increasingly diffused bands were seen with age in normally aging neurons. However, a typical nucleosomal ladder, characteristic of apoptosis, was seen only when the cells were exposed to either glutamate or MNNG irrespective of the age of the neurons. Furthermore, this apoptotic fragmentation of DNA was prevented by prior treatment of the cells with either cycloheximide or aurintricarboxylic acid, indicating that both glutamate and MNNG induce programmed cell death. Fluorescence microscopic observation of glutamate- and MNNG-treated neurons after acridine orange staining revealed a high degree of staining and marked condensation of nuclear DNA. On the other hand, no such phenomenon was observed in normally aging neurons either histologically or in biochemical assays of damage. It is concluded that both glutamate and MNNG induce programmed cell death in neurons independent of age and that accumulation of DNA damage in naturally aging neurons occurs through a process other than that of apoptosis.