To our knowledge, ours is the first study to examine the activity of glutathione antioxidant enzymes and the levels of protein oxidation and tyrosine nitration in patients in different stages of bipolar disorder. Our results indicate that patients in the late stage of illness show increased activity of GR and GST and increased levels of 3-nitrotyrosine compared with healthy controls, whereas patients in the early stage demonstrate only increased 3-nitrotyrosine levels. Protein oxidation, verified by carbonyl levels, and GPx activity did not differ among patients in the early- or late-stage groups and matched controls. These results indicate that nitration of tyrosine residues occur early in the course of bipolar disorder.
There is evidence that 3-nitrotyrosine can cause inflammation.44
Our previous studies have shown that patients in the early and late stages of bipolar disorder have elevated levels of proinflammatory cytokines interleukin-6 and tumour necrosis factor-α, whereas the anti-inflammatory cytokine interleukin-10 was increased only in patients in the early stage of the disorder.45
This suggests that anti-inflammatory defence mechanisms are no longer operational in late-stage bipolar disorder. With increased duration of illness, it is likely that the cumulative effect of ongoing oxidative stress results in elevation of antioxidant enzymes (GR and GST) as a compensatory mechanism to reduce further oxidative damage and illness progression among patients with bipolar disorder. In this sense, if the compensatory/antioxidants mechanisms do not react in time, these patients’ conditions may progressively worsen. This suggests an important role for compensatory antioxidant mechanisms in illness progression in patients with bipolar disorder. However, it is likely that such compensatory mechanisms are only partially effective, as previous studies have shown that patients with bipolar disorder exhibit increased levels of oxidative damage to proteins, lipids and DNA18–27
as well as reduced brain-derived neurotrophic factor (BDNF) levels.45
Our findings are in line with recent models that highlight the importance of early intervention in bipolar disorder.46,47
The delay in the initiation of suitable treatment strategies early in the course of bipolar disorder can exert substantial functional damage,46,47
as indicated by reduced BDNF levels and increased oxidative damage in patients in the late stage of bipolar disorder. Indeed, currently available treatments for bipolar disorder, such as lithium and divalproex, have proven antioxidant effects48
and thus have the potential to minimize oxidative damage and prevent further deterioration in the course of bipolar disorder.
If oxidative stress is present in bipolar disorder, what might be responsible for this? There is evidence that increased DA levels are associated with the symptoms of mania and that a reduction in DA transmission through reduction in DA synthesis or blockade of DA D2
receptors may be associated with antimanic effects.49,50
Interestingly, increased DA levels are an important source of oxidative stress in the brain owing to oxidative metabolism of DA.51,52
Dopamine may be metabolized through monoamine oxidase with production of hydrogen peroxide and dihydroxyphenylacetic acid53,54
or through nonenzymatic hydroxylation in the presence of ferrous ion and hydrogen peroxide leading to the formation of 6-hydroxydopamine (6-OHDA).11,55
6-Hydroxydopamine is toxic to the nervous system, and the mechanisms involved in this toxicity include endoplasmic reticulum stress, activation of glycogen synthase kinase-3-β by phosphorylation at tyrosine 216 and inhibition of the protein kinase AKT by phosphorylation at Ser473.14
In-vivo electrochemical measurements have shown that within a few minutes after injection into a rat brain, about 20% of 6-OHDA is converted to p-quinone,14,15
which can also be conjugated with glutathione by GST.6,12
This reaction is similarly thought to combat degenerative processes in the dopaminergic system in the human brain.56
Moreover, Tirmenstein and colleagues,57
using human neuroblastoma cells, showed that after 4 hours of treatment, 6-OHDA substantially depleted cellular and glutathione concentrations, whereas after 24 hours it induced a concentration-dependent increase in glutathione and total glutathione concentrations, suggesting that 6-OHDA induces oxidative stress in SH-SY5Y cells resulting in an adaptive increase in cellular glutathione concentrations. Our results demonstrate that only patients in the late stage of bipolar disorder have increased levels of GR and GST (). In addition, GR and GST levels show positive correlations with duration of illness (). The increase in antioxidant enzymes in patients in the late stage of bipolar disorder may be a consequence of cumulative effect of oxidative stress with progression of bipolar disorder.
Neuroimaging studies have shown increased levels of glutamate+glutamine and lactate levels in discrete subregions of the prefrontal cortex in adult patients with bipolar disorder.58
-methyl-D-aspartate receptors are ionotropic glutamate receptors that, when stimulated by glutamate, allow the passage of calcium into the cell, promoting activation of calcium/calmodulin-dependent protein kinase type IV, which may activate nitric oxide synthases (NOS1 and NOS3), thereby leading to increased nitric oxide production. Supporting this scenario, increased nitric oxide levels have been demonstrated in patients with bipolar disorder.22,23,59
In addition, increased intracellular calcium levels are a consistent finding in studies of bipolar disorder.60
Nitric oxide can react with superoxide, leading to the formation of peroxynitrite, which has the ability to nitrosylate free tyrosine and tyrosine residues in proteins, forming 3-nitrotyrosine.61
Our results indicate that patients in the early and late stages of bipolar disorder have increased levels of 3-nitrotyrosine (). The modification of critical cellular proteins by peroxynitrite-induced tyrosine nitration has been proposed as an early event in the process of DA neuronal damage.62
In addition, Ji and Bennet17
showed that microsomal GST is activated on exposure to peroxynitrite by nitration of tyrosine residues. The activation of GST by peroxynitrite may play an important role in limiting the extent of oxidative tissue injury when other cellular antioxidant enzymes such as superoxide dismutase63
and xantine oxidase64
are compromised under pathophysiological conditions of excessive peroxynitrite formation. Given that 3-nitrotyrosine is commonly measured as a biomarker of reactive nitrogen species generation,65
our results suggest that the nitration process may play an important role in the pathophysiology of bipolar disorder.
Recent findings have suggested that mitochondrial dysfunction may be associated with the pathophysiology of bipolar disorder.66
Studies conducted in postmortem brain tissue have demonstrated that mitochondrial genes are downregulated in the hippocampi67
and dorsolateral prefrontal cortices of patients with bipolar disorder,68,69
whereas superoxide dismutase and catalase expression is decreased in the hippocampi of patients with bipolar disorder.70
may promote inhibition of the complex I of the mitochondrial electron transport chain. In addition, Ferger and colleagues72
suggest that 6-OHDA can increase protein nitration levels. In our view, reactive nitrogen species may be a link between DA-related oxidative stress and mitochondrial dysfunction. Future studies will need to investigate the levels of 6-OHDA and its relation to increased reactive nitrogen species in patients with bipolar disorder.
Some limitations should be considered when interpreting the results of our study. First, we used a cross-sectional design and therefore could only examine associations between the glutathione enzymes activity and protein damage, but not direct causative mechanisms or the effects of progression of illness. Second, the cohort included patients from different regions of the world; however, we reduced the potential confounding effect by matching patients with controls in the same region by sex, age and education. Nevertheless, we cannot exclude the nonspecific environmental bias on the direct comparisons between patient groups. Third, the patients included in our study were taking psychotropic medication, thus our results could reflect effects of chronic medication use. We found a positive association between GST activity with the use of antidepressants or mood stabilizers. Previous studies involving primary cultured neuronal cells have shown that lithium and valproate (first-line mood stabilizers) can increase the mRNA and protein levels of the GST M1 isoenzyme73
and that valproate inhibits FeCl3–induced lipid peroxidation and protein oxidation.48
In addition, Frey and colleagues74
have shown that lithium and valproate exert protective effects against amphetamine-induced oxidative stress in vivo. Long-term use of antidepressants (e.g., desipramine, imipramine, maprotiline, mirtazapine) increased the mRNA levels of glutathione-S-transferase and glutathione reductase in human monocytic U-937 cells,75
and atypical antipsychotics may counteract some of the progressive deteriorative effects by enhancing synaptic plasticity and cellular resilience.76