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Page 44 Bennett. J Transl Genet Genom 2020;4:36-49 I https://doi.org/10.20517/jtgg.2020.17

Chronic exposure to ROS elicits an additional response. Catalase expression is known to protect against
oxidative stress, and catalase overexpression protects against ROS-mediated cellular damage [121-124] . Because
[79]
catalase is not constitutively expressed at EPR-detectable levels in most tissues (liver is an exception) , the
appearance of catalase ferriheme EPR signals at g = 6.2 and 5.7, that flank the signal at g = 6, provides
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an oxidative biomarker for sustained exposure to ROS. This signal may not be of use for monitoring MD
progression, as it is for characterizing tumor growth, but could be a useful tool for evaluating therapy.
Where EPR can be much more powerful than when applied as a stand-alone tool for the characterization
of MD and in subsequent therapy evaluation is when it is integrated into a comprehensive multi-technique
protocol. The first and most comprehensive study of this type was the investigation of the underlying
mechanism of mitochondrial dysfunction in two deoxyguanosine kinase (DGUOK; EC 2.7.1.113)-deficient
[79]
rat models (“M1” and “M2”) of a genetic mitochondrial DNA depletion syndrome . Mitochondrial DNA
assays, histology, protein immunoblot (western blot) assays, and electron transport chain activity assays
were carried out in addition to EPR measurements. Both rat models were characterized by a 90% depletion
of hepatic mtDNA and a 60%-80% depletion of splenetic mtDNA, similar to those observed in the human
condition for which the model was developed [125] . One of the models, M2, was ~50% deficient in brain
mtDNA while the other, M1, was ~50% deficient in mtDNA in the quadriceps muscle overall although the
deficiency appears not to be uniformly distributed throughout the muscle. Histological staining revealed
numerous fibers in DGUOK-deficient rat muscle that were negative for Complexes II and IV whereas
no such fibers were evident in wild-type (w/t) rat muscle. MRC protein expression assays indicated that
Complexes II, IV, and V were expressed at w/t levels whereas Complexes I and III were expressed at about
50% of w/t. Activity assays of the MRC complexes, though, differed considerably from their expression levels.
Complex II activities in both liver and muscle of DGUOK-deficient rats were close to w/t levels, as was
that of Complex IV in muscle. Complexes I and III, however, exhibited very low activities, 10%-20% of w/t,
lower than expected from protein expression assays. Interestingly, the activity of Complex IV in liver was
< 20% of w/t despite being expressed at w/t levels. The EPR results were, therefore, considered in light of
these observations.

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EPR of liver indicated that the signals due to reduced [2Fe2S] and [4Fe4S] clusters of Complex I were
diminished by about 50% in DGUOK-deficient rats, regardless of the cluster midpoint potential. That the
same ratio of intensities of signals was observed from each of N1b, N2, N3, and N4 in w/t and MD rats
indicates that the redox potential experienced by Complex I was not affected, and that the signal diminution
in MD rat liver is therefore due to the proportionally diminished expression level. This result also indicates
that the lower-than-expected activity of Complex I is not due to globally misfolded protein or an inability
to incorporate at least the four EPR-characterized low-potential iron-sulfur clusters, but likely involves
the mitochondrially-encoded ND1 subunit. The other potentially interesting results of EPR of liver were a
2+
25% increase in the aconitase signal, and a doubling of a signal due to Mn . The aconitase signal suggests
ongoing oxidative stress, perhaps related to the presence of the fully-reduced but partially inactive Complex
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I. The expression of signals due to Mn has been associated with oxidative stress adaptation in bacteria [126] ,
but any role in humans is undefined. The only clear signals associated to either of Complexes III or IV were
+
the reduced Rieske [2Fe2S] cluster signal of Complex III, and the Complex IV heme a signal at g = 3. The
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Rieske cluster signal from MD rats exhibited the same intensity as w/t despite lower expression and even
lower activity. This observation is rationalized by the observation that Complex II is fully expressed and fully
functional in MD rat liver and that electron redistribution to Complex I is thermodynamically unfavorable
due to reduction of the latter. Therefore, Complex II is likely to be feeding reducing equivalents into a
depleted pool of dysfunctional Complex III, ensuring complete reduction of the Rieske cluster and leading
to a 50% depletion of the ferriheme a signal. The conclusion was the inability of fully-reduced Complex I
to donate its electrons into the MRC may render it the source of ROS, while a fully functional Complex II
is sufficient to support reduction of Complex III which, due to the low activities of Complexes III and IV