ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1862-1882 © Pleiades Publishing, Ltd., 2025.
1862
REVIEW
Suppressing Mitochondrial ROS Production
is Beneficial in Multiple Preclinical Models
of Human Disease
Martin D. Brand
Buck Institute for Research on Aging, 8001 Redwood Blvd., Novato, California 94945, USA
e-mail: mbrand@buckinstitute.org
Received August 12, 2025
Revised September 24, 2025
Accepted September 29, 2025
Abstract— I discuss the therapeutic potential of site-specific suppressors of the production of mitochondrial
reactive oxygen species (ROS). The best-defined suppressors are S1QELs (targeting site I
Q
in complex I) and
S3QELs (targeting site III
Qo
in complex III). They prevent ROS formation at source without affecting oxida-
tive phosphorylation. The antidiabetic drug imeglimin and the anti-xerostomia and antischistosomal anethole
dithiolethiones also have S1QEL activity, although how much this contributes to their clinical effects needs
further study. Suppressing mitochondrial ROS production has therapeutic potential in many diseases. S1QELs
and imeglimin improve glucose tolerance, insulin sensitivity, and decrease hepatic steatosis in models of
diabetes and obesity. S1QELs and S3QELs protect against age-related cardiac decline, atrial fibrillation and
hypertension. They reduce inflammatory cytokines and oxidative stress in macrophages and other cells. They
inhibit cancer cell proliferation and tumour growth. In neurological diseases, S1QELs protect against noise-in-
duced hearing loss. S1QELs protect against cardiac and hepatic damage during ischemia-reperfusion. S1QELs
and S3QELs extend lifespan in model organisms and S3QELs protect against aging-related intestinal barrier
dysfunction. Suppressors mitigate drug-induced toxicities (e.g., acetaminophen, cisplatin) and the effects of
environmental stressors. In exocrinopathy, anethole dithiolethione alleviates symptoms of dry mouth and dry
eye. Suppressors of mitochondrial ROS production show promise in treating a wide range of diseases driven
by mitochondrial oxidative stress. Their mechanism-based specificity offers advantages over traditional antiox-
idants, with potential applications in metabolic, cardiovascular, inflammatory, neurological, and aging-related
diseases. Further research is needed to fully explore their clinical efficacy.
DOI: 10.1134/S0006297925602527
Keywords: mitochondria, ROS, S1QEL, S3QEL, imeglimin, anethole dithiolethione, type 2 diabetes, cardiomy-
opathy, inflammation, cancer, noise-induced hearing loss, ischemia/reperfusion injury, aging, acetaminophen,
cisplatin
MITOCHONDRIAL ROS PRODUCTION
The production of reactive oxygen species (ROS)
by mitochondria has been studied extensively by
many laboratories for more than 50 years [1-17].
It has become clear that there are at least 11 dif-
ferent sites within the electron transport chain and
associated dehydrogenases of mammalian mitochon-
dria at which electrons can leak and cause premature
reduction of molecular O
2
to form the two primary
ROS: superoxide (by one-electron reduction of O
2
)
and hydrogen peroxide (by two-electron reduction)
[5, 6, 18-28].
Which mitochondrial sites form ROS at the fast-
est rates in the matrix and in the cytosol in intact
cells (and, by extension, in vivo)? Using isolated rat
muscle mitochondria incubated in a medium mim-
icking the cytosol of muscle cells at rest or during
exercise, four sites were found to dominate, two in
respiratory complex I (sites I
Q
and I
F
) and one each
in respiratory complexes II (site II
F
) and III (site III
Qo
)
[29, 30], see Fig. 1.
SUPPRESSING MITOCHONDRIAL ROS PRODUCTION IN PRECLINICAL MODELS OF DISEASE 1863
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 1. Major sites of ROS production associated with substrate oxidation and the mitochondrial electron transport chain
in mitochondria exvivo and in cells. Red circles indicate the four major sites of superoxide/hydrogen peroxide production
that have been implicated in isolated rat muscle mitochondria ex vivo under conditions mimicking those in vivo; two of
these sites (sites I
Q
and III
Qo
) dominate mitochondrial ROS production in isolated cells. The energetics of electron flow are
illustrated by three planes representing different groups of redox centres. Centres within each group operate at about the
same redox potential (E
h
), indicated by the right-hand scale. Electrons are passed from metabolites to the electron trans-
port chain by dehydrogenases, shown as ovals, and normally flow within an isopotential group and then down to the next
isopotential group, until they reach oxygen at E
h
~ +600 mV, reducing it to water. As electrons drop from one isopotential
group to the next at respiratory complexes I, III and IV, protons are pumped across the mitochondrial inner membrane,
and energy is conserved as protonmotive force (pmf). Electrons enter the NADH/NAD
+
pool through NAD-linked dehy-
drogenases including the 2-oxoglutarate dehydrogenase complex (OGDHC) and pyruvate dehydrogenase complex (PDHC),
which can leak electrons to generate superoxide/hydrogen peroxide (sites O
F
and P
F
respectively), although they have yet
to be shown to do so in cells or in vivo. During forward electron transport (FET) electrons from NADH flow into the fla-
vin-containing site of complex I (site I
F
) then descend via the quinone-binding site (site I
Q
) to the next isopotential pool.
Dehydrogenases linked to ubiquinone (Q) also pass electrons into the QH
2
/Q pool, particularly complex II (site II
F
) and
mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH, site G
Q
). Electrons from QH
2
are passed to the outer Q-bind-
ing site of complex III (site III
Qo
), then to centre III
Qi
and down through cytochrome c and complex IV to oxygen. During
reverse electron transport (RET) electrons from QH
2
are driven thermodynamically uphill to the NADH/NAD
+
isopotential
group by pmf (generated by FET through complexes III and IV, or by ATP hydrolysis), and feed into sites I
Q
and I
F
to gener-
ate superoxide/hydrogen peroxide. The sites of action of suppressors of superoxide/hydrogen peroxide production (S1QELs
at site I
Q
and S3QELs at site III
Qo
) are marked.
Within a wide range of intact cultured mamma-
lian cells provided with different respiratory sub-
strates, two of these sites turned out to be quantita-
tively most important; sites I
Q
and III
Qo
[31-34]. Site
I
Q
is the site in respiratory complex I that is routine-
ly measured during reverse electron transport (RET)
from ubiquinol to NAD [5, 6, 24, 35, 36], although it
generates ROS equally well during forward electron
transport (FET) from NADH to ubiquinone when elec-
tron transport is stalled under resting conditions [37].
(The distinction between ROS production during FET
and RET that is sometimes drawn in the literature
is not helpful – what matters is not the net direc-
tion of electron flow but the levels of the factors
that cause high ROS production from site I
Q
, namely
high QH
2
/Q and NADH/NAD
+
ratios, high membrane
potential, and independently high pH gradient [9, 24,
37, 38]). In intact cells site I
Q
dominates superoxide
and hydrogen peroxide production in the mitochon-
drial matrix, producing about 70% of the total in that
compartment [31-33]. Matrix superoxide is rapidly
converted to hydrogen peroxide by superoxide dis-
mutase-2, and hydrogen peroxide derived from site I
Q
then spills out into the cytosol and makes up about
15% of total cytosolic ROS production. Site III
Qo
is the
outer quinone-binding site in respiratory complex III
[5, 6, 22]; it delivers superoxide to both the mitochon-
drial matrix and the cytosol [3, 22]. In intact cells site
III
Qo
is the other major mitochondrial contributor to
ROS production in the mitochondrial matrix and in
the cytosol, providing about 30% of the total in each
case; much of the remaining cytosolic hydrogen per-
oxide (40-60%) comes from cytosolic NADH oxidases
(NOXs) [31-33].
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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 2. Outline structures of the ROS suppressors discussed in this review. See the original references for their stereo-
chemistry where appropriate. Metformin is not known to be a S1QEL but is included to show its structural relationship
to imeglimin.
SUPPRESSING MITOCHONDRIAL ROS PRODUCTION IN PRECLINICAL MODELS OF DISEASE 1865
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
SITE-SPECIFIC SUPPRESSORS
OF MITOCHONDRIAL ROS PRODUCTION
Figure 2 shows the structures of the compounds
discussed in this review.
Diphenyleneiodonium is an NAD(P)H oxidase in-
hibitor that also potently inhibits mitochondrial ROS
production [49]. Following our demonstration that
diphenyleneiodonium specifically suppressed super-
oxide and hydrogen peroxide production by site I
Q
in isolated mitochondria without strongly inhibiting
forward electron transport and respiration [35], we
developed a plate-based screen for such suppressors
at several different sites in the mitochondrial electron
transport chain [39]. This was a mechanism-based
screen: we first set up conditions in isolated mito-
chondria under which only a single site of electron
leak produced a fluorescent signal, for example ROS
production specifically from site I
Q
was measured as
the rotenone-sensitive increase in resorufin fluores-
cence in the presence of succinate to provide elec-
trons to respiratory complex I and generate proton-
motive force through respiratory complexes II, III
and IV [6, 39, 50]. We then screened for molecules
that strongly inhibited the signal from one site with-
out affecting other sites or respiration and oxidative
phosphorylation. Importantly, this screen rejects can-
didates that have general antioxidant activity or that
inhibit normal electron transport and energy metab-
olism, although one set of such rejects turned out to
be useful novel inhibitors of mitochondrial glycerol
phosphate dehydrogenase, iGP-1 and iGP-5 [51]. Using
this screen on a small chemical library we identified
a better suppressor of ROS production by site 1
Q
:
N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide,
CN-POBS [39]. Scaling up of the screen to a much larg-
er chemical library of about a million compounds led
to the identification of several independent classes of
S3QELs (suppressors of site III
Qo
electron leak) [43]
and S1QELs (suppressors of site I
Q
electron leak) [40].
In our hands these early compounds were powerful
tools to investigate the importance of sites I
Q
and
III
Qo
in isolated mitochondria [29, 30, 36, 37, 40, 43],
in cells [30-33, 40, 43, 52], in isolated perfused organs
[40], and (to a limited extent because of bioavailabili-
ty) in vivo [34, 40, 41, 52, 53]. Subsequent application
of medicinal chemistry led to improved S1QELs, such
as S1QEL1.719, with high affinity (the free concentra-
tion giving half-maximal suppression (IC
50
) is about
50 nM) and better solubility and pharmacokinetics,
enabling them to be given orally to animals to study
the effects of suppression of ROS production by mi-
tochondrial site I
Q
in preclinical models in vivo [42].
S1QEL1.719 has suitable solubility, permeability and
metabolic stability for oral administration dosed as
a suspension in 0.5% w/v hydroxypropyl methylcellu-
lose. Total unbound exposures in mice measured 2 h
after oral gavage of 30 mg S1QEL719/kg body weight
were about 100 nM in plasma and about 150 nmo-
l/g in liver. These exposure values (near C
max
) were
2-3-fold greater than the IC
50
for suppression of super-
oxide/H
2
O
2
production from site I
Q
and 200-300-fold
less than the IC
50
of 30 µM for inhibition of respira-
tion on complexI substrates, determined using isolat-
ed rat skeletal muscle mitochondria [42]. In addition,
S1QEL1.719 is well-tolerated, with no adverse effects
noticed at these exposures after 40 weeks of supple-
mentation to chow. Similarly, mice have been given
high doses of S3QEL2 in chow for over 12 months with
no detectable adverse effects on health, body weight,
metabolism, or general behavior, and S3QEL2 readily
crossed the gut and blood-brain barriers, leading to a
brain to plasma ratio of about 2 [54]. Further rounds
of primary screening and medicinal chemistry have
since generated structurally unrelated new “next-gen-
eration” S1QELs with excellent drug-like properties;
these work in vivo after oral administration at low
doses and have displayed no off-target effects; in par-
ticular they show no inhibition of electron transport
and oxidative phosphorylation even at much higher
doses. Authentic S1QELs and S3QELs have not yet
been submitted for approval for use in humans, so
all current information about their potential clinical
efficacy comes from studies in preclinical cell and
animal models as detailed below. The mechanism
by which they suppress ROS production is not estab-
lished, but we can exclude the suggestion that S1QELs
work by inhibiting reverse electron transport into
complex I [36]. Our working hypothesis is that they
alter rate constants within complex I and complex III
respectively, lowering the steady-state concentration
of the electron donor (presumably a semiquinone)
that reacts with O
2
to generate superoxide or hydro-
gen peroxide, without affecting the rates of forward
or reverse electron transport through the complexes.
Site-specific suppressors of mitochondrial ROS
production are inherently different from antioxidants,
even those specifically targeted to mitochondria, be-
cause suppressors prevent the formation of superox-
ide and hydrogen peroxide at their source, whereas
antioxidants lower the concentrations of these ROS or
interfere with their downstream effects. An analogy
may help to illustrate this difference: if mitochondrial
ROS production is a bottle of red wine tipped on its
side, and the rest of the cell and organism are the
expensive white carpet below, then antioxidants mop
up the spilled wine and try to limit its spread, where-
as suppressors of mitochondrial ROS production keep
the bottle stoppered to prevent the damage from oc-
curring in the first place.
Other compounds are also known to act as
S1QELs, having a demonstrated ability to suppress ROS
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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
production by site I
Q
: imeglimin [55, 56] and aneth-
ole dithiolethione (anethole trithione) [57]. Imeglimin
[44] (brand name Twymeeg), derived from metformin
by addition of acetaldehyde [45], is an oral antidia-
betic drug that has been successful in phase III trials
[58, 59] and is now used for the treatment of type II
diabetes in Japan [60] and India [61, 62]. It has clear
S1QEL activity [55], albeit needing very high concen-
trations (100 µM in digitonin-permeabilised human
endothelial cells [56], and, in our hands, specificity
for site I
Q
but a very poor IC
50
of only about 2 mM
in isolated rat muscle mitochondria using our stan-
dard assays [5, 6, 20, 39, 40]. However, the molecular
mechanisms of its therapeutic action are thought to
be complex and are not fully understood [55, 61, 63,
64]. As well as decreasing mitochondrial ROS genera-
tion, it affects mitochondrial function by partial inhi-
bition of complex I and stimulation of complex III ac-
tivity, prevents mitochondrial permeability transition
pore opening, improves insulin sensitivity, modulates
genes involved in hepatic gluconeogenesis, and pro-
tects beta-cell function with induction of the synthe-
sis of NAD via the salvage pathway. Anethole dithio-
lethione (5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione,
ADT, also known as anethole trithione, AOL, Felviten,
Sialor, and OP2113) and closely related compounds
such as oltipraz (4-methyl-5-(2-pyrazynyl)-1,2-dithiole-
3-thione) have been used in humans for many de-
cades; they improve bile production by the liver to
aid digestion, improve saliva production in cases of
dry mouth, act as antischistosomal agents and have
chemoprotective effects against cancers and xenobi-
otics [65-70]. Anethole dithiolethione has clear S1QEL
activity, albeit with low potency, with an IC
50
of
10-26µM [57, 71]. It is reasonably specific; IC
50
values
against other mitochondrial sites of ROS production
are at least 15-fold higher. It did not affect oxidative
phosphorylation in isolated mitochondria or respira-
tion or growth of C2C12 cells [71]. However, the mo-
lecular mechanisms of its therapeutic actions remain
unclear; in particular, it is a known slow-release H
2
S
donor [47, 70, 72].
DO IMEGLIMIN AND ANETHOLE
DITHIOLETHIONE ACT SIGNIFICANTLY
AS S1QELs, OR DO THEIR PRECLINICAL
AND CLINICAL EFFECTS DEPEND PARTLY
OR COMPLETELY ON OTHER TARGETS?
There is an important conceptual difference be-
tween the S1QELs (and S3QELs) on the one hand,
and imeglimin and anethole dithiolethione on the
other: mechanism-based versus phenotypic screening.
Imeglimin was developed using an in vivo phenotypic
screen of antihyperglycaemic activity in rodents, fol-
lowed by chemical modification of a lead molecule
[61, 64]. Anethole dithiolethione has been known for
many years from its effects on complex human phe-
notypes. Thus, the preclinical and clinical actions of
imeglimin and anethole dithiolethione are not disput-
ed, but the specific molecular target or targets un-
derlying their clinical effects are open for test and
discussion. They do have (weak) S1QEL activity, but
their clinical effects might be caused partially or com-
pletely by other, separate molecular mechanisms yet
to be fully delineated, particularly bearing in mind
their poor potencies as S1QELs as discussed above.
On the other hand, S1QELs (and S3QELs) were discov-
ered by mechanism-based primary screening to find
specific suppressors of ROS production at sites I
Q
and
III
Qo
respectively, with counterscreens for any effects
at other sites or on electron transport and oxidative
phosphorylation, as discussed above. This screening
yielded several completely different chemical fami-
lies of compounds (S1QEL1, S1QEL2 etc., see Fig. 2)
each with the same mechanistic ability to suppress
ROS production at site I
Q
, but otherwise unrelated.
Ifeach of these chemically unrelated S1QELs is found
to have the same preclinical or clinical effect in any
particular case, that provides strong evidence that
the effect is caused specifically by the suppression of
superoxide/hydrogen peroxide production at site I
Q
,
which is the defining feature of all S1QELs, and not
to some unknown off-target effect, which would be
specific for one or another specific class (S1QEL1 but
not S1QEL2, etc.). The same logic applies separately to
the different classes of S3QELs: if each of the chem-
ically unrelated S3QELs is found to have the same
preclinical or clinical effect in any particular case,
that is strong evidence that the effect is caused spe-
cifically by the suppression of superoxide/hydrogen
peroxide production at site III
Qo
, which is the defin-
ing feature of all S3QELs, and not to some unknown
off-target effect, which would be specific for one or
another specific class (S3QEL1 but not S3QEL2, etc.).
We have reported such similarity of action in mech-
anistic and preclinical models for chemically unrelat-
ed S3QELs [30-32, 43, 53] and chemically unrelated
S1QELs [30-32, 36, 37, 40, 41]. We have also routine-
ly run S1QEL1.719 [42] alongside structurally unre-
lated “next-generation” S1QELs and seen the same
effects with each in many of our preclinical models
discussed below, confirming that their preclinical ef-
fects are on-target. In this way, the molecular target
of S1QELs (and S3QELs) is not in significant doubt,
but their preclinical and clinical actions are open for
test and discussion. A case can be made that many of
the beneficial clinical effects of imeglimin and aneth-
ole dithiolethione are each primarily caused directly
or indirectly by their ability to act as S1QELs to sup-
press mitochondrial ROS production at site I
Q
, thereby
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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
sparing mitochondrial and cellular glutathione, de-
creasing lipid peroxidation, and protecting from in-
flammation. If so, their clinical effects are an excel-
lent guide to the potential clinical efficacy of S1QELs
in general, and the effects of S1QELs discussed below
are an excellent guide to potential new indications
against which imeglimin or anethole dithiolethione
should be effective, and a guide to how tweak their
structures to improve their efficacy. However, if the
mechanism of imeglimin or of anethole dithiolethi-
one is partly or completely through other, non-S1QEL,
effects then imeglimin and anethole dithiolethione
make poor or misleading guides to the clinical ac-
tions expected of more clearly-defined S1QELs, which
would then be in a mechanistically separate class.
Agood way to find out whether or not the preclinical
and clinical effects of imeglimin or anethole dithio-
lethione arise solely through their action as S1QELs
is to compare their effects with those of validated
S1QELs. Any discrepancies would be caused by non-
S1QEL effects of imeglimin or anethole dithiolethi-
one. Conversely, complete overlap of effects would be
strong evidence that they work exclusively as S1QELs.
WHAT CLINICAL INDICATIONS
ARE LIKELY TO BE IMPROVED
BY TREATMENT WITH SUPPRESSORS
OF MITOCHONDRIAL ROS PRODUCTION?
Oxidative stress is thought to have a role in many,
or even most, diseases and pathologies. Untangling
primary causes from secondary consequences, and
the extent to which mitochondrial ROS production
from specific sites may drive disease initiation and
progression, requires careful attention to the type of
evidence that is available. Many published studies are
associative rather than mechanistic; they show that
some signal attributed to mitochondrial ROS (such
as the expression of an antioxidant protein, or the
response of a fluorescent probe) is increased when
the physiological signalling pathway or pathology is
activated, or decreased when it is inactive. Such as-
sociations are suggestive, but they do not distinguish
between causal and bystander or downstream roles
of mitochondrial ROS, and are therefore not defini-
tive. The following criteria for assessing whether mi-
tochondrial production of superoxide and/or hydro-
gen peroxide drives biological or pathological effects,
ranked by reliability, have been proposed [7].
1. Inhibition of a phenotype by addition of well-vali-
dated S1QELs or S3QELs indicates that it is driven
by superoxide or hydrogen peroxide generated at
mitochondrial site I
Q
or site III
Qo
respectively.
2. Inhibition of a phenotype by overexpression of
mitochondria-specific superoxide dismutase-2
(SOD2) to lower the superoxide concentration in
the mitochondrial matrix, or, conversely, exac-
erbation of the phenotype by raising matrix su-
peroxide level by SOD2 deficiency, indicates that
the phenotype is driven by superoxide in the
matrix.
3. Inhibition of a phenotype by overexpression of
peroxiredoxin-3 (PRDX3) or by expression of syn-
thetic mitochondrially-targeted catalase (mCAT),
to lower matrix hydrogen peroxide level, or, con-
versely, exacerbation of the phenotype by raising
matrix hydrogen peroxide level by PRDX3 defi-
ciency, indicates that the phenotype is driven by
matrix hydrogen peroxide.
4. Inhibition of a phenotype by mitochondria-tar-
geted antioxidants that react specifically with
superoxide (and hydrogen peroxide), such as
mitoTEMPO, to lower matrix levels of superoxide
(and hydrogen peroxide) suggests that the pheno-
type is driven by matrix superoxide (or hydrogen
peroxide).
5. Inhibition of a phenotype by mitochondria-target-
ed antioxidants, such as mitoQ, SkQs, and SS-31,
which interfere with downstream lipid peroxi-
dation, suggests that the phenotype is driven by
matrix ROS, although the exact targets and mech-
anisms then require further elucidation.
6. Studies associating a pathology with human gene
variants in relevant proteins (SOD2 or PRDX3)
suggest that the human pathology is driven or
exacerbated by matrix superoxide or hydrogen
peroxide respectively.
Using these criteria to sift the huge literature
on the role of oxidative stress in disease provides
strong and compelling evidence that mitochondrial
ROS are causal or sensitizing for a wide range of
human pathologies [7]. These pathologies overlap
but can be crudely grouped into the categories listed
in Table 1.
For each of these broad and overlapping pathol-
ogies, there is strong evidence from all or most of
the six criteria enumerated above that mitochondrial
ROS production is a primary driver or sensitizer of
the pathology. In particular, knockout of mitochon-
drial SOD-2 to raise matrix superoxide concentration
exacerbates the phenotype in many models of dis-
ease, and matrix expression of catalase to decrease
matrix hydrogen peroxide concentration or treatment
with the mitochondrially-targeted spin trap mito-
TEMPO protects (see [7] for the relevant references
to the primary literature). Mitochondrially-targeted
lipid peroxidation chain-breakers such as mitoQ [73]
and SkQ [74] have been particularly effective; stud-
ies using them in preclinical models and in patients
are fully referenced elsewhere [7]. These studies by
Murphy and colleagues and by Skulachev and col-
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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Table 1. Pathologies for which there is a range of strong and compelling evidence that mitochondrial ROS
are causal or sensitizing [7], and *evidence for beneficial effects of treatment with S1QELs or S3QELs (or the
putative S1QELs imeglimin and anethole dithiolethione) in preclinical models of these diseases; for details see
the text
Pathology S1QEL S3QEL Imeglimin
Anethole
dithiolethione
A Metabolic disease (diabetes, obesity) * * * *
B Cardiovascular diseases (hypertension, atherosclerosis,
cardiomyopathy, heart failure)
*** *
C Inflammatory diseases (inflammation, atherosclerosis) * *
D Cancer (incidence, growth, metastasis) * * *
E Neurological diseases (cognitive, motor, degenerative,
vision, hearing, neuropathies)
(i) Dementia *
(ii) Noise-induced hearing loss *
(iii) Parkinson’s disease * *
F Ischemia/reperfusion injuries * * *
G Aging and associated diseases * *
H External insults
(i) Tunicamycin and ER stress * *
(ii) Acetaminophen hepatotoxicity * * *
(iii) Carbon tetrachloride hepatotoxicity *
(iv) Cisplatin nephrotoxicity *
(v) Fluoroquinolone tenotoxicity *
I Genetic diseases
(i) POLG mouse *
J Other
(i) Exocrinopathy *
leagues have not only identified and characterized
therapeutic targets for such antioxidants themselves,
but have also illuminated areas in which suppressors
of ROS production might be effective. It follows from
these examples that multiple pathologies should be
amenable to treatment or amelioration by suppres-
sion of mitochondrial ROS production. In this review
I focus on the extent to which this expectation has
been explored and tested using known mitochondri-
al ROS suppressors: S1QELs and S3QELs, and, with
the strong caveat discussed above regarding the ex-
tent to which they may or may not act purely as
S1QELs, using imeglimin and anethole dithiolethiones.
See Table 1 for a summary.
METABOLIC DISEASES
There is strong empirical evidence from the ad-
ministration of S1QELs (and of imeglimin and aneth-
ole dithiolethione) that mitochondrial ROS production
in vivo is a driver of metabolic disease and that its
suppression can ameliorate the pathology.
S1QEL. Knockout of SOD2 in mice raises matrix
superoxide concentration, and leads to hepatic steato-
sis, a classic symptom of metabolic syndrome. SOD2
–/–
mice have dilated cardiomyopathy, accumulation of
lipid in liver and skeletal muscle, metabolic acidosis,
a failure of weight gain and a median lifespan of
8 days [75]. Dosing of Sod2
−/−
mouse pups with early
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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
S1QELs that were suitable for intraperitoneal injec-
tion but not for oral administration (S1QEL1.712 or
S1QEL2.352) led to decreased hepatic steatosis (but
dosing with S3QEL1.941 did not) [41]. A classic way
to model metabolic syndrome is acute 1-2-week or
longer-term 8-week high-fat-feeding of mice, which
leads to insulin resistance [76]. In such mice a more
advanced S1QEL1 that is suitable for oral dosing
(S1QEL1.719) decreased fat accumulation, improved
glucose tolerance and normalized fasting insulin con-
centration both prophylactically and therapeutically
[42], showing that ROS production from mitochon-
drial site I
Q
in vivo is necessary for the induction
and maintenance of glucose intolerance caused by a
high-fat diet in mice and suggesting that oral admin-
istration of S1QELs may be beneficial in metabolic
syndrome. Our more recent studies have replicated
and amplified these results using “next-generation”
S1QELs with single or multiple prophylactic or ther-
apeutic doses of S1QEL1.719, and shown that dosing
with S1QEL1.719 in this model results in increased
glucose uptake in tissues in a hyperinsulinemic-eu-
glycemic clamp assay and lowers plasma levels of
the cytokines fibroblast growth factor 21 (FGF21) and
growth differentiation factor 15 (GDF15), which are
elevated by cellular stress in diet-induced obesity [77].
We have also shown that therapeutic or prophylac-
tic oral dosing of S1QEL1.719 and “next-generation”
S1QELs in mice fed a methionine-choline deficient
diet (a mouse model of non-alcoholic steatohepati-
tis and fibrosis [78, 79]) improves glucose tolerance
and protects against hepatic damage, steatosis and
fibrosis. We have also found that S1QEL1.719 dosing
lowers circulating insulin levels in genetic models of
metabolic disease: db/db and ob/ob mice. Supporting
evidence comes from studies showing that application
of S1QEL2.2 decreased ROS production in mitochon-
dria isolated from the livers of obese mice more than
in those from control mice; in hepatocytes from obese
mice use of S1QEL2.2 showed that expression of the
alternative ubiquinol oxidase (AOX) decreased site I
Q
ROS production, allowing the beneficial effect of AOX
in vivo in mice to be interpreted as due to decreased
site I
Q
ROS production. AOX-expressing mice had low-
er fasting blood glucose levels, improved intraperito-
neal and oral glucose tolerance and decreased liver
glycogen content than controls [80].
S3QEL. S3QEL2 protects isolated pancreatic beta
cells and insulinoma INS-1E cells against oxidative
stress ex vivo [43, 81], but dosing of Sod2
−/−
mouse
pups with S3QEL1.941 did not lead to decreased he-
patic steatosis [41], suggesting that superoxide produc-
tion from site III
Qo
is only an important source of ROS
damage in beta cells under specific circumstances.
Imeglimin. Imeglimin improved the three key
pathological defects of diabetes in streptozotocin-
treated rats, namely excessive hepatic glucose pro-
duction, impaired peripheral glucose uptake by skel-
etal muscle, and insufficient insulin secretion [44].
Six-week imeglimin treatment had antidiabetic effects
in a 16-week high-fat, high-sucrose diet mouse model
of metabolic disease. It normalized glucose tolerance
and insulin sensitivity by preserving mitochondri-
al function from oxidative stress and favoring lipid
oxidation in liver [55]. Imeglimin directly activated
beta-cell insulin secretion in awake rodents [82].
It improved mitochondrial function, reduced hepatic
steatosis, and suppressed hepatic fibrosis in mice fed
a choline-deficient high-fat diet [83]. In patients with
type 2 diabetes, imeglimin improved beta-cell function
[84]. Recent reviews document the clinical efficacy of
imeglimin in decreasing steatosis and improving beta
cell function and glycemic control in type 2 diabetes
[59, 62, 85]. The effects of S1QELs in preclinical mod-
els partly mirror those of imeglimin, so a case can be
made that the beneficial clinical effects of imeglimin
in treatment of type 2 diabetes and metabolic disease
are partly or even completely explained by its action
as a weak S1QEL, although this remains a speculation
that needs to be tested.
Anethole dithiolethione. Dithiolethione com-
pounds such as oltipraz can prevent or treat fibrosis
and insulin resistance, and have mitochondrial pro-
tective effects in the liver, by a mechanism proposed
to involve AMP-activated protein kinase (AMPK) and/
or 70-kDa ribosomal protein S6 kinase 1 (S6K1) [69].
Oltipraz improved blood glucose and insulin resis-
tance, decreased blood lipid metabolism, reduced in-
flammation and apoptosis, suppressed oxidative stress,
mitigated pancreatic and liver tissue injury, and en-
hanced pancreatic beta-cell insulin secretion, thereby
mitigating the symptoms of type 2 diabetic mice [86].
A novel mitochondria-targeted anethole dithiolethione
H
2
S donor (AP39) decreased hyperglycemia-induced
oxidative stress and metabolic changes in microvas-
cular endothelial cells in vitro. Targeting H
2
S to mito-
chondria using AP39 induced a 1000-fold increase in
the potency of the cytoprotective effect of H
2
S against
hyperglycemia-induced injury, suggesting that AP39
could be useful against diabetic vascular complica-
tions [47]. The respective authors attribute the poten-
cy of oltipraz to its effects on the AMPK–mTOR–S6K1
pathway [69] and the effects of AP39 to its activity as
an H
2
S donor in the mitochondrial matrix [47, 87].
The extent to which these compounds may have been
acting more directly as S1QELs remains undetermined.
CARDIOVASCULAR DISEASES
There is some empirical evidence from the ad-
ministration of S1QELs, S3QELs, imeglimin and
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anethole dithiolethione that mitochondrial ROS pro-
duction in vivo is a driver of atrial fibrillation, car-
diomyopathy and hypertension and that its suppres-
sion can ameliorate these pathologies.
S1QELs and S3QELs. In isolated rabbit atrial
myocytes, S1QEL1.1 and S3QEL2 decreased the sever-
ity of beat-to-beat alternations in atrial calcium tran-
sient amplitude, which are causally linked to atrial
fibrillation [88]. We found that long-term feeding of
S1QEL1.719 in chow (starting at 12 weeks of age and
finishing at 54 weeks of age) had no obvious detri-
mental effect on C57BL/6J mice. Echocardiography of
54-week old mice fed S1QEL1.719 for 42 weeks re-
vealed that they were significantly protected against
the normal age-related decline in cardiac function,
specifically ejection fraction, stroke volume, cardiac
output and end-diastolic volume.
Imeglimin. Imeglimin improved cardiac gene
expression abnormalities associated with heart fail-
ure with preserved ejection fraction in mice subject-
ed to the cardiometabolic stress of high-fat diet and
the nitric oxide synthase inhibitor L-NAME for 16
weeks [89].
Anethole dithiolethione. Anethole dithiolethione
decreased contractile hyperreactivity to 5-hydroxy-
tryptamine and prostaglandin F2α in pulmonary ar-
terial rings from chronic hypoxia-induced pulmonary
hypertension rats [90]. In vivo, preventive treatment
with anethole dithiolethione decreased mean pulmo-
nary arterial pressure, pulmonary artery remodelling
and right ventricular systolic pressure and reversed
pulmonary artery hyperreactivity to 5-hydroxytrypt-
amine [91, 92].
INFLAMMATORY DISEASES
(INFLAMMATION AND ATHEROSCLEROSIS)
Suppressors of mitochondrial ROS production
are effective at decreasing inflammatory cytokines in
various contexts, although they have yet to be tested
widely in overt models of atherosclerosis and other
inflammatory diseases.
S1QEL. S1QEL1.1 decreased the production of the
pro-inflammatory cytokines tumour necrosis factor-α
(TNF-α) and interleukin-1b in mouse macrophages
exposed to swollen conidia of Aspergillus fumigatus,
and reduced the fungicidal activity of macrophages
against swollen conidia [93]. Treatment with a S1QEL
suppressed lipopolysaccharide-induced expression of
interleukin-10 and augmented that of interleukin-6 in
cultured macrophages under serine deprivation [94].
S1QEL treatment decreased oxidized low-density lipo-
protein-induced glycolytic reprogramming, levels of
pro-inflammatory cytokines interleukin-1b and CXCL8
(formerly interleukin-8), and foam cell formation in
isolated human monocytes [95]. In our hands thera-
peutic S1QEL1.719 treatment strongly attenuated ex-
pression of a panel of inflammatory genes in liver of
mice fed a methionine-choline deficient diet.
S3QEL. Treatment with S3QEL2 decreased mouse
bone marrow macrophage TNF-α and IFN-β levels
following lipopolysaccharide stimulation and TNF-α,
CXCL10, IFN-α and IFN-β production following
Poly(I:C)-activation [96]. In a murine alveolar mac-
rophage cell line incubated with particulate matter,
treatment with a S3QEL inhibited interleukin-6 pro-
duction [97]. Treatment with a S3QEL suppressed li-
popolysaccharide-induced expression of interleukin-10
and augmented that of interleukin-6 in cultured mac-
rophages under serine deprivation [94]. S3QEL2 de-
creased ATPγS-induced production of interleukin-6
in human airway epithelial cells [98]. S3QEL1.2 de-
creased interleukin-10 in lipopolysaccharide-activated
macrophages and impaired interleukin-10 production
in vivo after lipopolysaccharide challenge [52].
CANCER
S1QELs and S3QELs can suppress cell prolifer-
ation and differentiation in a variety of non-cancer-
ous and cancerous cell types. A S1QEL attenuated
normoxic proliferation of mouse lung epithelial cells
[99]. S1QEL1.1 and S3QEL2 impaired myogenesis
during C2C12 myoblast differentiation [100]. S3QEL3
suppressed the cross-presentation capacity of activat-
ed plasmacytoid dendritic cells to elicit a clonal CD8
+
T cell expansion response [101]. S3QEL2 reduced the
growth of Jurkat cells subjected to respiratory inhi-
bition by antimycin or piericidin [102]. A S3QEL de-
creased proliferation of Drosophila intestinal stem
cells following Ecc15 infection [103]. A S3QEL, but not
a S1QEL, protected mouse hepatoma cells from lipid
peroxidation and the subsequent ferroptosis induced
by cysteine starvation, and suppressed ferroptosis in
xCT-knockout mouse-derived embryonic fibroblasts,
which usually die under conventional cultivating con-
ditions due to the absence of intracellular cysteine
and glutathione [104].
As well as these in vitro effects, S3QELs and
anethole dithiolethiones have shown effectiveness
against carcinoma in vivo.
S3QEL. S3QEL1.2 promoted tumour-mediated im-
mune evasion and promoted survival of mice bearing
B16F10 melanoma by lowering tumour growth [52].
Anethole dithiolethione. Anethole dithiolethi-
one inhibited colon carcinogenesis [105], significant-
ly inhibited mammary cancer multiplicity [106] and
is a potentially efficacious chemoprevention agent
for lung cancer [68, 107]. It inhibited anchorage-
independent growth of A549 cells, inhibited the
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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
migration of A549 cells in the transwell assay and
inhibited lung cancer cell proliferation and decreased
tumour growth almost 2-fold in an orthotopic mouse
xenograft model with A549 human lung adenocar-
cinoma cells in vivo [108, 109]. Oltipraz has proved
effective as an inhibitor of carcinogenesis in exper-
imental models of breast, bladder, liver, forestom-
ach, colon, tracheal, lung, and skin cancer; mecha-
nistic studies indicate that it affects the metabolism
and disposition of chemical carcinogens, principally
through the induction of electrophile detoxication
enzymes [46, 69].
NEUROLOGICAL DISEASES
(DEMENTIA, NOISE-INDUCED HEARING LOSS,
PARKINSON’S DISEASE)
Some observations suggest that suppression of
mitochondrial ROS production can be protective
against neurological stresses and diseases, although
more work is needed to show this definitively.
(i) DEMENTIA
S3QEL. Chronic administration to mice of high
doses of S3QEL2 in chow for over 12 months had no
detectable adverse health effects and did not alter
body weight, metabolism, or general behavior. In this
study S3QEL2 treatment decreased dementia-linked
tauopathy and neuroimmune cascades and extended
lifespan [54]. The authors suggest that site-specific
suppression of ROS generated at site III
Qo
represents
a promising therapeutic intervention in dementia and
other neurological conditions that affect mitochondri-
al ROS production.
(ii) MOISE-INDUCED HEARING LOSS
S1QEL. We have found that treatment of mice
with S1QEL1.719 gave significant protection (by about
10dB in the threshold for sound perception) against
permanent noise-induced hearing loss, was cytopro-
tective to inner hair cells in animals subjected to
noise at 32 kHz and to outer hair cells in animals
subjected to noise at 8 kHz, and protected ribbon
synapses in animals subjected to noise at 32 kHz.
S3QEL1.941 [41] did not protect.
(iii) PARKINSON’S DISEASE
S1QEL. Treatment with S1QEL1.1 attenuated mi-
tochondrial ROS production and cell death induced
by MPP
+
(the neurotoxin 1-methyl-4-phenylpyridini-
um) in SHSY5Y neuroblastoma cells (a cellular model
of Parkinson’s disease) [110]. Also, S1QEL1.1 co-ex-
posure rescued rotenone-induced dendritic degener-
ation and dopaminergic function in the roundworm
Caenorhabditis elegans [111]. However, this result
should be treated with caution, since rotenone pre-
vents ROS production at site I
Q
, so S1QEL co-expo-
sure should have had no further effect. Also, S1QELs
can decrease rotenone binding to complex I [36] and
might have been working by attenuating the initial
complex I inhibitory effect of rotenone (and perhaps
of MPP
+
).
S3QEL. Treatment with S3QEL2 attenuated mito-
chondrial ROS production and cell death induced by
MPP
+
in SHSY5Y neuroblastoma cells [110].
ISCHEMIA/REPERFUSION
INJURIES
There is robust evidence that suppressing ROS
production from site I
Q
is protective against isch-
emia-reperfusion injury.
S1QEL (and S3QEL). Therapeutic administration
of S1QEL1.1 was found decrease ROS production by
site I
Q
in mouse muscle mitochondria and to pro-
tect against ischemia-reperfusion injury in the per-
fused mouse heart [40], providing strong supporting
evidence that ROS production from site I
Q
is a pri-
mary cause of cardiac ischemia-reperfusion injury.
Wehave repeated and extended such observations us-
ing S1QEL1.719 and unrelated new “next-generation”
S1QELs in mouse and rat hearts. Injection of a S1QEL
during cardiopulmonary resuscitation in KCl-cardiac
arrest mice improved myocardial function, neuro-
logic outcomes, and survival and represents a po-
tential therapy for improving sudden cardiac arrest
resuscitation outcomes [112, 113]. Pretreatment with
S1QEL1.1 suppressed the immediate increase in the
fluorescence of mitoSOX, a ROS indicator, at the on-
set of reperfusion after ischemia in isolated mouse
hearts [114], and S1QEL1.1 treatment limited infarct
size in isolated perfused hearts from UCP3-knockout
mice subjected to ischemia-reperfusion ex vivo [115].
A hydrogel incorporating S1QEL1.1 and other agents
significantly improved cardiac function and reduced
adverse ventricular remodelling in an in vivo rat
model of myocardial ischemia-reperfusion injury
[116]. A strong decrease in ROS production from site
I
Q
caused by addition of S1QEL1.1 was confirmed us-
ing isolated mouse heart mitochondria under condi-
tions simulating ischemia-reperfusion injury ex vivo,
and a small contribution of superoxide production
from site III
Qo
preventable by S3QEL2 was also seen
under more constrained conditions [117, 118].
Anethole dithiolethione. Anethole dithiolethione
(OP2113) improved recovery of contractile activity and
decreased infarct size in a rat heart infarct model,
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although the authors did not rule out the possibility that
the beneficial effects were due instead to complex I in-
hibition or the activity of anethole dithiolethione as
an H
2
S donor [57]. Anethole dithiolethione decreased
ST segment elevation, decreased troponin release,
improved left ejection fraction and decreased infarct
size in a sheep model of regional ischemia-reperfu-
sion [71] and decreased myocardial infarct size and
no reflow after rat myocardial ischemia-reperfusion
[119, 120]. An anethole dithiolethione prodrug with
improved solubility (ATXP) maintained the bioactiv-
ity of the parent drug (5-(4-hydroxyphenyl)dithiole-
3- thione) to cause a significant reduction in infarct
volume 24 h after reperfusion [48].
AGING AND AGING-ASSOCIATED DISEASES
Aging is a dominant risk factor for many of the
diseases discussed in the present review; the benefi-
cial effects of suppressing mitochondrial ROS produc-
tion in many of them are treated separately in the
other sections (see metabolic diseases, cardiovascular
diseases, inflammatory diseases, cancer, dementia,
Parkinson’s disease). S1QELS and S3QELs also have
protective effects on intestinal barrier dysfunction
(which can limit lifespan in roundworms) and lifes-
pan, although more work is needed to test their an-
ti-aging properties and the relationships between the
beneficial effects of mitochondrial ROS-suppressors
on aging-related diseases and any effects on aging
itself.
S1QEL. We have found that S1QEL1.719 and un-
related “next-generation” S1QELs extend median lifes-
pan in C. elegans [unpublished].
S3QEL. S3QEL1.2, S3QEL2.2, and S3QEL3 protect-
ed against greater intestinal permeability and against
shortened lifespan on high-nutrient diets in Drosoph-
ila, and S3QEL1.2 and S3QEL2.2 strongly protected
against greater diet-induced intestinal permeability
in mice [53].
EXTERNAL INSULTS
(DRUG-INDUCED PATHOLOGIES –
TUNICAMYCIN, ACETAMINOPHEN,
CARBON TETRACHLORIDE, CISPLATIN,
FLUOROQUINOLONES)
Several drug-induced pathologies are caused, at
least in part, by depletion of endogenous glutathione,
particularly in liver. The resulting damage caused by
unchecked endogenous production of ROS leads to
oxidative stress, causing overt drug-induced pathol-
ogies that may be lessened by prophylactic adminis-
tration of mitochondrial ROS suppressors.
(i) TUNICAMYCIN AND ENDOPLASMIC
RETICULUM STRESS
Tunicamycin inhibits the N-linked oligosaccha-
ride formation of glycoproteins in the endoplasmic
reticulum and triggers the protective unfolded pro-
tein response. High tunicamycin dosage may saturate
the unfolded protein response, leading to hepatic ste-
atosis [121, 122]. We speculate that the resulting cal-
cium imbalance in the endoplasmic reticulum leads
to calcium uptake into the mitochondria, activating
calcium-sensitive dehydrogenases of the tricarboxylic
acid cycle and causing increased ROS production by
the electron transport chain, triggering S1QEL- and
S3QEL-sensitive downstream effects such as hepato-
steatosis.
S1QEL. Administration of S1QEL1.1 and S1QEL2.2
protected against caspase3 and 7 cleavage in embry-
onic cardiomyocyte H9C2 cells treated with tunica-
mycin, showing that ROS production by site I
Q
is in-
volved in the endoplasmic reticulum stress signalling
pathway in these cells [40]. We found that S1QEL1.1
and S1QEL1.719 also protected these cells against tu-
nicamycin-induced apoptosis. Dietary S1QEL1.1 and
S1QEL2.2 protected against tunicamycin stimulation
of intestinal cell proliferation in vivo in Drosophila
[40]. In human hepatic cells (AML12), we have found
that S1QEL1.719 ameliorates tunicamycin-induction of
the PERK-eIF2a-ATF4-CHOP-FGF21 signalling pathway.
In mice, we have found that intraperitoneal injection
of S1QEL1.712 gives significant protection against tu-
nicamycin-induced body weight loss and tunicamy-
cin-induced hepatosteatosis measured by oil red O
staining, and oral administration of a “next-genera-
tion” S1QEL attenuates the increase in FGF-21 caused
by tunicamycin administration [unpublished].
S3QEL. S3QEL1, S3QEL2, and S3QEL3 protected
against tunicamycin-induced cleavage of caspase 3
and 7 in a rat INS-1 insulinoma cell line [43] and
S3QEL2.1 protected against tunicamycin-induced
cleavage of caspase 3 and 7 in embryonic cardiomyo-
cyte H9C2 cells [40]. S3QEL1.2, S3QEL2 and S3QEL3
decreased apoptosis and necrosis in tunicamycin-
treated C2C12 myoblasts [123]. In vivo, we have found
that intraperitoneal injection of S3QEL1.941 [41] de-
creases liver fat deposition measured using oil red O
in tunicamycin-treated mice.
(ii) ACETAMINOPHEN HEPATOTOXICITY
High doses of acetaminophen lead to the forma-
tion of reactive metabolites of acetaminophen in the
liver. These cause oxidative stress and hepatotoxicity
by depletion of glutathione and inhibition of mitochon-
drial glutathione peroxidases, removing some of the
protection against damage by endogenous ROS [124].
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S1QEL. Leakage of the liver-specific enzyme al-
anine aminotransferase into the bloodstream is a
marker of liver cell damage. We have found that
S1QEL1.712 and S1QEL2.1 protect against alanine
aminotransferase leakage from acetaminophen-treat-
ed HepG2 cells, and that prophylactic intraperitone-
al injection of S1QEL1.712 and oral administration
of low doses of S1QEL1.719 and many unpublished
S1QEL2s and “next-generation” S1QELs strongly pro-
tect against acetaminophen-induced leakage of al-
anine aminotransferase activity into the plasma of
mice in vivo. They also protect against elevated plas-
ma levels of the mitochondrial stress-related cytokine
GDF15. These observations raise the possibility that
prophylactic or simultaneous application of S1QELs
may be effective against acute acetaminophen toxic-
ity in humans.
S3QEL. In vivo, we have found that intraperi-
toneal injection of S3QEL1.941 [41] decreased plas-
ma alanine aminotransferase activity in acetamino-
phen-treated mice.
Anethole dithiolethione. Administration of dithi-
olethiones to mice protected against the acute toxic
effects of acetaminophen [125-127].
(iii) CARBON TETRACHLORIDE
HEPATOTOXICITY
The mechanism of carbon tetrachloride hepato-
toxicity is complex, although antioxidants can be pro-
tective [128].
Anethole dithiolethione. Administration of dithi-
olethiones and their derivatives to mice protected
against the acute toxic effects of carbon tetrachloride
[125, 129, 130].
(iv) CISPLATIN NEPHROTOXICITY
Cisplatin is a widely-used and effective anti-can-
cer agent, but its use is limited by kidney damage.
Accumulation of cisplatin in renal tubular cells caus-
es DNA damage, mitochondrial pathology, oxidative
stress and endoplasmic reticulum stress [131]. Lower-
ing oxidative stress by suppressing mitochondrial ROS
formation may be protective against kidney injury.
S1QEL. We have found that that prophylactic
oral administration of S1QEL1.719 and “next-genera-
tion” S1QELs potently protect mice against cisplatin
renal toxicity as measured by blood urea nitrogen
(BUN) and plasma levels of creatine and the specific
kidney damage marker KIM1.
(v) FLUOROQUINOLONE TENOTOXICITY
Fluoroquinolone antibiotics inhibit bacterial DNA
replication and are the most intensively applied an-
tibiotics in both human and veterinary medicine.
However, they can cause side effects such as tendon
damage, thought to be mainly related to ROS and ox-
idative stress [132].
Anethole dithiolethione. Anethole dithiolethione
administration was found to decrease the level of
ROS induced by fluoroquinolone antibiotics in teno-
cytes [133].
GENETIC DISEASES
(i) POLG MOUSE
Mitochondrial DNA mutations underlie sever-
al genetic diseases. One model to study them is the
POLG mutator mouse, which carries a proof-read-
ing-deficient version of mtDNA polymerase and has
a shortened lifespan and premature onset of ageing-
related phenotypes [134]. Although it is controversial,
some authors argue that ROS and oxidative stress
contribute to the phenotype [135].
S1QEL. We have found that 30 weeks of dietary
S1QEL1.719 improves latency to fall on the rotarod in
POLG mutator mice at 42 weeks of age.
OTHER
(i) EXOCRINOPATHY (HYPOSALIVATION,
DRYMOUTH, XEROSTOMIA,
SJÖGREN SYNDROME, DRY EYE,
XEROPHTHALMIA)
Anethole dithiolethione. The historical clinical
application of anethole dithiolethione is in the treat-
ment of hyposalivation. Anethole dithiolethione in-
creased salivary secretion from the rat submaxillary
gland induced by electrical stimulation of the para-
sympathetic nerve and by injection of pilocarpine
[136] and may enhance salivary secretion by stimu-
lating the postjunctional secretory process involved
in the parasympathetic nervous system [65, 137]. In
patients with autoimmune exocrinopathy (Sjogren’s
syndrome) anethole dithiolethione alleviated the
symptoms of xerostomia [138]; several other authors
have found it to have beneficial effects in treating xe-
rostomia [67, 139, 140] and xerophthalmia [138, 141].
CONTRAINDICATIONS
Although the superoxide and hydrogen peroxide
generated by mitochondria cause cellular damage and
drive pathologies [7], they are also used as signals to
drive appropriate physiological responses [142, 143].
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Suppressors of mitochondrial ROS production will
not only ameliorate damage and disease, but also
decrease ROS signalling. However, much of this sig-
nalling promotes appropriate cellular responses to
the initial cellular insults that would be caused by
ROS production, particularly activation of signalling
through protein kinases such as mitogen-activated
protein kinase (MAPK) and phosphoinositide 3-ki-
nase (PI3K)/protein kinase B (Akt), and through tran-
scription factors including nuclear factor erythroid
2-related factor 2 (Nrf2), hypoxia-inducible factor 1α
(HIF-1α), activator protein 1 (AP-1), and nuclear fac-
tor kappa-light-chain-enhancer of activated B cells
(NF-κB) [143]. To the extent that these pathways are
activated only to counter the potentially harmful ef-
fects of mitochondrial ROS production, then their de-
crease by ROS-suppressors would be simply a harm-
less reflection of the fact that they are not needed
when mitochondrial ROS production is decreased.
ROS signalling for purposes other than defense
against mitochondrial ROS production is a different
matter. Acute hypoxic pulmonary vasoconstriction
(HPV) may be such an exception. Humans and other
animals respond to low alveolar oxygen levels with
acute hypoxic pulmonary vasoconstriction to divert
blood flow to better-oxygenated parts of the lung, and
with increased ventilation driven by chemoreceptors
in the carotid bodies, which detect changes in blood
oxygen levels and signal the brain to increase venti-
lation. Conversely, at birth oxygen rises, and the duc-
tus arteriosus constricts to shunt blood to the lungs.
Mitochondria in glomus cells of the carotid body, in
small pulmonary artery smooth muscle cells, and in
ductus arteriosus smooth muscle cells are accepted to
be the oxygen sensors. In these cells, changes in oxy-
gen tension rapidly alter production of mitochondrial
ROS, which in turn regulate the opening of redox-sen-
sitive potassium channels, altering plasma membrane
potential, calcium influx through voltage-gated calci-
um channels, and smooth muscle contraction [144].
A number of papers argue that these responses are
driven by ROS produced from mitochondrial sites
I
Q
or III
Qo
, although different groups favor different
sites [144-148]. Use of different inhibitors of the elec-
tron transport chain, including diphenyleneiodonium
(which also has S1QEL activity [35]), and antioxidants
in model systems (buffer-perfused rat lungs, pulmo-
nary artery myocytes) suggested that ROS generated
in the proximal region of the electron transport chain
(complex I) act as second messengers in HPV [145].
Mitochondrial complex III was required for hypox-
ia-induced ROS production and cellular oxygen sens-
ing [149], and this requirement in cells was narrowed
down to ROS production by site III
Qo
[147]. This con-
clusion was supported by the effects of genetic knock-
out of the activity of the Rieske iron-sulphur protein
of complex III in pulmonary artery myocytes, and
ex vivo [146] whereas the involvement of site I
Q
was
supported by the effects on HPV of knockdown of the
complex I subunit NDUFS2 in carotid body cells and
in vivo [144, 150, 151]. The use of inhibitors of elec-
tron transport and selective knockdown of electron
transport chain subunits in these studies is very prob-
lematic, as these manipulations alter not only ROS
production, but also ATP production and the redox
states of the mitochondria and cytosol independently
of ROS [7], so the use of selective suppressors of ROS
production (S1QELs and S3QELs) that do not change
electron transport is preferred.
A S1QEL was found to reverse oxygen-induced
constriction in rabbit ductus arteriosus rings, and
in human ductus arteriosus smooth muscle cells it
inhibited oxygen-induced increases in cytosolic cal-
cium, a surrogate for ductus arteriosus constriction
[144, 152]. Therefore, the use of S1QELs immediately
before and at birth deserves attention as a potential
contraindication. However, we have found that oral
administration of S1QEL1.719 has no significant effect
on either baseline ventilation or the acute hypoxic
ventilatory response before or after 48 h acclimati-
zation to hypoxia in awake, unrestrained mice using
individual whole-body plethysmographs, providing
no support for a S1QEL contraindication in HPV in
adults.
S3QEL2 was found to attenuate hypoxia-induced
plasma membrane depolarization and HPV in human
pulmonary arterial smooth muscle cells [153], and a
S3QEL inhibited HPV in isolated mouse lungs [154].
In contrast, a S3QEL did not inhibit oxygen-induced
ductus arteriosus constriction exvivo and in vivo [152].
CONCLUSION
Suppressors of mitochondrial ROS production
show promise in treating a wide range of diseases
driven by mitochondrial oxidative stress. Their mech-
anism-based specificity offers great advantages over
traditional antioxidants, with potential applications
in metabolic, cardiovascular, inflammatory, neurolog-
ical, and aging-related diseases. Further research is
needed to fully explore their clinical efficacy and any
potential contraindications.
Funding
This work was supported by ongoing institutional
funding. No additional grants to carry out or direct
this particular review were obtained.
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects.
SUPPRESSING MITOCHONDRIAL ROS PRODUCTION IN PRECLINICAL MODELS OF DISEASE 1875
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Conflict of interest
The author of this work declares that he has no con-
flicts of interest.
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