This is the first in-depth study of the regulation of mitochondrial PTP opening and antioxidant-related mitochondrial proteins by a pharmacologic activator of the Nrf2 pathway of cytoprotective gene expression. The primary conclusion reached from this study is that treatment of rats with sulforaphane results in a robust inhibition of PTP opening in rat liver mitochondria triggered by several forms of oxidative stress. These forms include a shift in redox state induced through either glutathione-dependent peroxide metabolism or direct oxidation of pyridine nucleotides by NADH-dependent reduction of oxaloacetate. In addition, sulforaphane treatment also inhibits PTP opening by direct oxidation of sulfhydryl groups mediated by phenylarsine oxide. An additional novel finding from this study is that sulforaphane treatment selectively increases several important mitochondrial proteins and molecules that serve as direct antioxidants, e.g. glutathione peroxidase, thioredoxin, and glutathione, or that generate the reducing power in the form of NADPH that is necessary for driving these antioxidant activities, i.e., malic enzyme.
A number of findings provide insight into molecular mechanisms responsible for inhibition of PTP opening by treatment of rats with sulforaphane. The > 100% increase in NQO1 immunoreactivity in liver homogenates strongly suggests that sulforaphane activates the Nrf2 pathway of gene expression [45
]. While the pattern of mitochondrial protein expression that is elevated by sulforaphane treatment supports this conclusion, additional comparisons between mitochondria from Nrf2 +/+ and Nrf2 −/− mice will be necessary to prove that Nrf2 activation is necessary for sulforaphane inhibition of PTP opening [46
]. One mechanism of action that can be ruled out is a general influence of sulforaphane treatment on basic mitochondrial bioenergetics, based on no differences observed for rates of state 3 or state 4 respiration. These findings do not, however, address the possibility that sulforaphane and other Nrf2 activators can stimulate mitochondrial biogenesis, as described by Piantadosi et al. [47
The possibility that sulforaphane treatment inhibits PTP opening by reducing the expression of PTP components remains an open question, due mainly to the fact that the identity of these components is unresolved. We focused our measurements on CyD since the relationship between its expression and PTP activity is best characterized and because it has no apparent direct influence over mitochondrial bioenergetics like other putative PTP components, e.g., the adenine nucleotide transporter and the phosphate transporter. We found that sulforaphane treatment has no effect on CyD immunoreactivity in rat liver mitochondria, which is consistent with the lack of an effect reported earlier for rat brain mitochondria [34
Insight into mechanisms that are applicable to the inhibition of PTP opening by sulforaphane comes from comparisons between release of accumulated Ca2+
and oxidization of pyridine nucleotides at different concentrations of t
BOOH. Similar rates of t
BOOH-induced mitochondrial Ca2+
release and complete NAD(P)H oxidation were observed at 5, 50, and 250 μM t
BOOH for mitochondria from vehicle-treated rats (). While Ca2+
release rates and pyridine nucleotide oxidation were also similar at both 50 and 250 μM t
BOOH with mitochondria from sulforaphane-treated rats, both were much reduced at 5 μM t
BOOH and also much lower than the values obtained with mitochondria from vehicle-treated animals. This comparison is similar to what we reported when comparing redox- sensitive PTP activity for mitochondria for normal rat liver and AS-30D hepatoma mitochondria. Compared to normal liver mitochondria, hepatoma mitochondria are resistant to both t
BOOH-induced NAD(P)H oxidation and Ca2+
]. We determined that the cause for this resistance is increased mitochondrial malic enzyme activity, which produces NADPH and increases the intramitochondrial redox buffering power. The fact that ME3 immunoreactivity is more than twice as high in the liver mitochondria from rats treated with sulforaphane () is consistent with the redox buffering power being responsible for resistance to PTP opening, at least at the low t
BOOH concentration of 5 μM. Another possible explanation is that t
BOOH is metabolized so quickly that the redox state is only temporarily disturbed, resulting in only transient PTP opening. The transient rise in medium [Ca2+
] concentration and reduction in NAD(P)H autofluorescence described in for mitochondria from sulforaphane-treated rats is consistent with this mechanism. This hypothesis is also supported by the finding that the total peroxidase activity of these mitochondria is significantly greater than that of mitochondria from vehicle-treated rats ().
The increase in either malic enzyme or mitochondrial peroxidases could explain sulforaphane-induced resistance of PTP opening at low t
BOOH levels but is unlikely to be responsible at concentrations of 50 μM and above. At these higher t
BOOH levels, complete and sustained pyridine nucleotide oxidation occurs with mitochondria from both vehicle- and sulforaphane treated rats, thus overcoming any elevated rates of NADPH production or peroxide metabolism. The increase in mitochondrial total reduced glutathione observed after sulforaphane administration could contribute to increased sulfhydryl buffering power; however the 25% increase is modest (). Another possible mechanism may lie distal to redox buffering and be limited kinetically rather than thermodynamically by reduction of protein thiol groups. The increased thioredoxin immunoreactivity observed in mitochondria from sulforaphane-treated rats is consistent with this possibility (). There are several other proteins that could also contribute to maintenance of reduced sulfhydryl redox state for PTP associated proteins like CyD. These proteins include but are not limited to thioredoxin reductase, glutaredoxin and glutaredoxin reductase [49
In summary, measurements of peroxide induced release of accumulated Ca2+ and pyridine nucleotide oxidation demonstrate that redox-sensitive PTP opening by isolated liver mitochondria is substantially inhibited by treatment of rats with i.p. sulforaphane over one and a half days earlier, particularly at lower levels of added tBOOH that are likely most relevant to levels of oxidative stress that occur in situ within cells. Several mechanisms of action are probably responsible for this inhibition, including increased redox buffering power, more rapid disposition of added peroxide, and more effective use of mitochondrial redox buffering to maintain PTP associated protein sulfhydryls in the reduced redox state. Experiments are in progress to further characterize the relative contribution of each of these mechanisms in this system. Considering the over 100 genes whose expression are induced by Nrf2 activators like sulforaphane, it is not surprising that the mitochondrion, like the entire cell, responds in multiple ways to Nrf2 activation. This multifactorial response probably confers mitochondria with protection against different forms of oxidative stress and many sequelae, including PTP opening and associated metabolic dysfunction.