biomolecules

Article
Effect of mitoTEMPO on Redox Reactions in Different Body
Compartments upon Endotoxemia in Rats
Adelheid Weidinger 1,† , Andras T. Meszaros 1,2,† , Sergiu Dumitrescu 1 and Andrey V. Kozlov 1, *

1 Ludwig Boltzmann Institute for Traumatology, The Research Center in Cooperation with AUVA,
1200 Vienna, Austria
2 Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck,
6020 Innsbruck, Austria
* Correspondence: [email protected]; Tel.: +43-059393-41980
† These authors contributed equally to this work.

Abstract: Mitochondrial ROS (mitoROS) control many reactions in cells. Biological effects of mi-
toROS in vivo can be investigated by modulation via mitochondria-targeted antioxidants (mtAOX,
mitoTEMPO). The aim of this study was to determine how mitoROS influence redox reactions in
different body compartments in a rat model of endotoxemia. We induced inflammatory response by
lipopolysaccharide (LPS) injection and analyzed effects of mitoTEMPO in blood, abdominal cavity,
bronchoalveolar space, and liver tissue. MitoTEMPO decreased the liver damage marker aspartate
aminotransferase; however, it neither influenced the release of cytokines (e.g., tumor necrosis factor,
IL-4) nor decreased ROS generation by immune cells in the compartments examined. In contrast,
ex vivo mitoTEMPO treatment substantially reduced ROS generation. Examination of liver tissue
revealed several redox paramagnetic centers sensitive to in vivo LPS and mitoTEMPO treatment and
high levels of nitric oxide (NO) in response to LPS. NO levels in blood were lower than in liver, and
were decreased by in vivo mitoTEMPO treatment. Our data suggest that (i) inflammatory mediators
are not likely to directly contribute to ROS-mediated liver damage and (ii) mitoTEMPO is more likely
to affect the redox status of liver cells reflected in a redox change of paramagnetic molecules. Further
Citation: Weidinger, A.; Meszaros,
studies are necessary to understand these mechanisms.
A.T.; Dumitrescu, S.; Kozlov, A.V.
Effect of mitoTEMPO on Redox
Reactions in Different Body
Keywords: reactive oxygen species; systemic inflammatory response syndrome; cytokines;
Compartments upon Endotoxemia in mitochondria-targeted antioxidants; bronchoalveolar system; peritoneal lavage; mitoTEMPO
Rats. Biomolecules 2023, 13, 794.
https://doi.org/10.3390/
biom13050794
1. Introduction
Academic Editors: José Pedraza
Chaverri, Mark Rinnerthaler and Systemic inflammation and consequent systemic inflammatory response syndrome
Markus Ralser (SIRS) is a complex cascade of pro- and anti-inflammatory processes. It is associated with
release of inflammatory mediators such as tumor necrosis factor (TNF)-α and interleukins
Received: 22 February 2023 (IL-1, IL-6, IL-4, MCP1 and others) [1] as well as by excessive generation of nitric oxide
Revised: 27 April 2023
(NO) by inducible nitric oxide synthase (iNOS), thought to be a mediator of distant organ
Accepted: 29 April 2023
damage [2]. The exact mechanism of cellular damage is not fully clarified, although the
Published: 5 May 2023
theory of a deleterious interplay of inflammatory mediators complemented by immune
cell cascade is widely accepted [1]. The question remains of the extent to which immune
cells and their products play a causative role in the distant organ damage.
Copyright: © 2023 by the authors.
The inflammatory response is often accompanied by multiple organ dysfunction syn-
Licensee MDPI, Basel, Switzerland. drome (MODS), which is often associated with excessive generation of reactive oxygen and
This article is an open access article nitrogen species (RONS). It has been shown that this process is orchestrated by mitochon-
distributed under the terms and drial ROS (mitoROS), mainly superoxide (O2•− ), a primary ROS generated by the electron
conditions of the Creative Commons transfer system in the mitochondrial inner membrane [3]. However, it is very difficult
Attribution (CC BY) license (https:// to dissect direct effects of ROS in vivo due to their very short lifetime. A common and
creativecommons.org/licenses/by/ precise way to access these biological effects in vivo is the examination of effects of so-called
4.0/). mitochondria-targeted antioxidants (mtAOX). These molecules comprise two structural

Biomolecules 2023, 13, 794. https://doi.org/10.3390/biom13050794 https://www.mdpi.com/journal/biomolecules

Biomolecules 2023, 13, 794 2 of 13

parts. The first is positively charged and hydrophobic. This part is considered as chemically
inert, and brings the entire molecule to the negatively charged mitochondrial matrix. The
second part has specific ROS-scavenging moieties [4]. TEMPO is the most often used
antioxidant part of the molecule.
Generally, the major reason for increased generation of mitochondrial ROS is a shift
in the redox equilibrium within the cells. These disturbances are often accompanied by
changes in intracellular redox active centers and deviation of electrons from their physio-
logical pathways (redox shuttles) to one-electron reduction of oxygen-yielding superoxide
radicals [5]. Such redox-motivated electron transfer appears in the mitochondrial elec-
tron transport chain (ETC) or electron transport system linked to cytochrome P450 in the
endoplasmic reticulum. One-electron reduction of oxygen appears under pathological
circumstances such as hypoxia or inflammation. The major sites of leakage of electrons in
mitochondria are Complex I and III of the electron transfer system [6]. Another important
component of the respiratory chain is ubiquinone (Q10), which exerts both pro- and antiox-
idant capacities [7]. A number of these centers containing enzymes of mitochondria, such
as P450, Q10, and iron, can be detected in a specific redox state by electron paramagnetic
resonance spectroscopy (EPR).
Spatially, there are two general sources of ROS that are considered potentially dam-
aging for tissues. Extracellular ROS are mainly generated by immune cells [8], while
intracellular ROS production leads to cell damage from intracellular sources [2]. Thus,
it can be expected that the antioxidant mitoTEMPO acts both in intra- and extra-cellular
environments owing to the almost ubiquitous presence of mitochondria.
To this end, the effects of mtAOX on ROS generation and on various redox processes
were evaluated in a lipopolysaccharide (LPS)-induced rodent SIRS model. LPS induces
acute systemic inflammatory response in rodents [9] and humans [10]. This model mirrors
certain aspects of septic shock in humans, though the correlation between rodent LPS
models and clinical septic shock is poor [11]. Nonetheless, this model has provided the
majority of mechanistic insights into systemic inflammation [10], such as the release of
cytokines [12], induction of apoptosis [13], signal transduction [14], and others.
In the present study, we set out to characterize ROS generation in three major patho-
logically relevant sites of oxidative stress: (1) in the intravascular compartment (blood),
(2) in the intraabdominal compartment, and (3) in the bronchoalveolar system. To the
best of our knowledge, there are no studies on the effects of mitoTEMPO in both body
compartments and in a reference tissue (liver) susceptible to inflammatory damage.

2. Materials and Methods

2.1. Chemicals
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless other-
wise noted.

2.2. Animals
The in vivo experiments were performed on male Sprague Dawley rats (250–300 g/
390–540 g; Animal Research Laboratories, Himberg, Austria/Charles River, Germany)
which were kept under controlled standard animal housing conditions at least for 7 days
prior to usage in experiments with free access to standard laboratory rodent food and
water. The rats with high weight were used in experiments first; small animals were
kept approximately 2 weeks longer in the animal research facility of the institute. All
interventions were conducted in compliance with the Guide for the Care and Use of
Laboratory Animals published by the National Institute of Health with approval from the
Animal Protocol Review Board of the city government of Vienna, Austria (no. M58003956-
2011-9, no. 815758/2014/16). To prevent unnecessary pain, Buprenorphin (Richter Pharma
AG, Wels, Austria, 0.05 mg/kg body weight) was injected subcutaneously at the time of
the LPS treatment and 10 h thereafter. At the end of experiments, rats were anesthetized by
inhalation of a mixture of 3% isoflurane and oxygen (Vapor 2000, Dräger, Austria).
 Wels, Austria, 0.05 mg/kg body weight) was injected subcutaneously at the time of the LPS
treatment and 10 h thereafter. At the end of experiments, rats were anesthetized by inha-
Biomolecules 2023, 13, 794 lation of a mixture of 3% isoflurane and oxygen (Vapor 2000, Dräger, Austria). 3 of 13

2.3. Lipopolysaccharide and mtAOX Treatment

Rats were injected
2.3. Lipopolysaccharide andwith
mtAOXlipopolysaccharide
Treatment (LPS) from Escherichia coli serotype
026:B6 (activity ≥ 500,000 EU/mg) at a dose of 2.5 mg/kg body weight dissolved in saline
Rats were injected with lipopolysaccharide (LPS) from Escherichia coli serotype 026:B6
(Fresenius Kabi, Graz, Austria). Samples were collected at 16 h after LPS injection. In a
(activity ≥ 500,000 EU/mg) at a dose of 2.5 mg/kg body weight dissolved in saline (Frese-
separate set of experiments (Figure 1), animals were divided into four groups. All rats
nius Kabi, Graz, Austria). Samples were collected at 16 h after LPS injection. In a separate
were
set ofinjected with the
experiments same1),
(Figure dose of LPSwere
animals (Escherichia
divided coli
intoserotype 026:B6,
four groups. All8 rats
mg/kg body
were in-
weight, activity ≥ 10,000 EU/mg) dissolved in saline. Control animals were injected
jected with the same dose of LPS (Escherichia coli serotype 026:B6, 8 mg/kg body weight, with
saline only.
activity Treatment
≥ 10,000 EU/mg)withdissolved
mitoTEMPO (50 nmol/kg)
in saline. Controlwas administered
animals intraperitoneally
were injected with saline
at 1 h before LPS treatment and 11 h after LPS treatment. The LPS solution
only. Treatment with mitoTEMPO (50 nmol/kg) was administered intraperitoneally was vortexed at
for 1 min and sonicated for 30 s before application, then injected in the penis
1 h before LPS treatment and 11 h after LPS treatment. The LPS solution was vortexed vein under
isoflurane anesthesia
for 1 min and sonicated(Vapor
for 302000, Dräger,
s before Austria) then
application, in volumes
injectedranging from vein
in the penis 0.5 tounder
0.75
mL.
isoflurane anesthesia (Vapor 2000, Dräger, Austria) in volumes ranging from 0.5 to 0.75 mL.

Figure 1. Experimental protocol of LPS and mitoTEMPO treatment followed by blood and tissue
Figure 1. Experimental protocol of LPS and mitoTEMPO treatment followed by blood and tissue
sampling. Treatment with mitoTEMPO was administered intraperitoneally at 1 h before LPS treatment
sampling. Treatment with mitoTEMPO was administered intraperitoneally at 1 h before LPS treat-
and 11 h after LPS treatment. Blood samples were collected at 0/2/4/8/16 h and tissue samples at
ment and 11 h after LPS treatment. Blood samples were collected at 0/2/4/8/16 h and tissue samples
16 h. h.
at 16
2.4. Sampling of Blood and Liver Tissue
2.4. Sampling of Blood and Liver Tissue
After a small skin cut was made, the left femoral artery was dissected and catheterized
using After
a 24aGA small
i.v.skin cut was
cannula (BDmade,
Neoflon,the left femoral
Becton artery was
Dickinson dissected
Infusion Therapyand AB,
catheter-
Hels-
ized using a 24 GA i.v. cannula (BD Neoflon, Becton Dickinson
ingborg, Sweden). Next, 10–12 mL of whole blood was collected in a 50 mL Falcon Infusion Therapy AB, Hel-
tube
singborg, Sweden).
prefilled with 200 µL Next, 10–12 mL
of sodium of whole
heparin (1000blood
IU/mL, wasGilvasan
collectedPharma
in a 50 GmbH,
mL Falcon tube
Austria)
prefilled with 200
for subsequent µL of sodium
processing and forheparin (1000part
the in vitro IU/mL,
of theGilvasan
study. Pharma GmbH, Austria)
for subsequent processing and for the in vitro
For determination of mononitrosyl–hemoglobin complex part of the study. (NO-Hb) levels, blood sam-
ples were taken into Minicollect tubes coated with lithium(NO-Hb)
For determination of mononitrosyl–hemoglobin complex heparin levels,
(Greiner blood sam-
Bio-One
ples were taken into Minicollect tubes coated with lithium ◦
GmbH, Kremsmünster, Austria). After centrifugation at 4 C and 1600× g for 10 min, heparin (Greiner Bio-One
GmbH,
plasma was Kremsmünster,
removed and Austria). After centrifugation
the erythrocyte at 4 °C and
pellet was collected in 1 1600× g for syringes.
mL plastic 10 min,
plasma was removed
Subsequently, erythrocyteandpellets
the erythrocyte pellet was
were shock-frozen collected
in liquid in 1 mL
nitrogen and plastic
stored at −80 ◦ C
syringes.
Subsequently, erythrocyte pellets were shock-frozen in liquid nitrogen and stored at −80
until EPR measurement.
°C until EPR measurement.
Following blood sampling, animals were euthanized by decapitation. Subsequently,
Following
the liver blood and
was excised sampling, animals
transferred were euthanized
immediately by decapitation.
to a beaker filled with ice Subsequently,
cold Ringer
the liver was excised and transferred immediately to a beaker filled
solution (Fresenius Kabi, Austria). After cooling, the liver was cut into small pieces with ice cold Ringer
on a
solution
Petri dish (Fresenius
placed onKabi, Austria).
ice. Tissue After to
samples cooling,
a volumethe of
liver
0.4was
mL werecut into small
filled in 1pieces on a
mL plastic
Petri dish shock-frozen
syringes, placed on ice.inTissue
liquidsamples
nitrogen,toanda volume
stored of −80mL
at 0.4 ◦ Cwere filled in
for further 1 mL plastic
measurements.
syringes,
Liver tissueshock-frozen
for measurementin liquidof nitrogen,
mitochondrialand stored at −80was
respiration °C forusedfurther measurements.
freshly.
Liver tissue for measurement of mitochondrial respiration was used freshly.
2.5. Measurement of Mitochondrial Respiration
2.5. Measurement
Respiratoryofparameters
Mitochondrial
of Respiration
mitochondria were monitored using high-resolution
respirometry
Respiratory(Oxygraph-2k,
parameters Oroboros Instruments,
of mitochondria were Innsbruck,
monitored Austria) as previously
using high-resolution
described [15]. Rat liver homogenate was incubated in buffer containing 105 mM KCl,
respirometry (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) as previously de-
5 mM KH PO , 20 mM Tris-HCl, 0.5 mM EDTA, and 5 mg/mL fatty acid-free
scribed [15]. Rat liver homogenate was incubated in buffer containing 105 mM
2 4 bovine
KCl, serum
5 mM
albumin (pH 7.2, 37 ◦ C). Respiration was stimulated by the addition of 5 mM glutamate
and 5 mM malate. Transition to State-3 respiration was induced by the addition of 1 mM
adenosine diphosphate. Oxygen consumption rates were obtained by calculating the nega-
tive time derivative of the measured oxygen concentration. The respiratory control ratio
was calculated by dividing State 3 respiration by State 2 respiration.
Biomolecules 2023, 13, 794 4 of 13

2.6. Electron Paramagnetic Resonance Spectroscopy

EPR is the most appropriate method to detect redox active centers directly in untreated
tissues. It can be used to determine redox-active iron-sulfur species [16], copper containing
compounds [17], free radicals and ferrous ions [18], and nitric oxide [19]. A particular
advantage of this method is that the analytic procedure can be performed directly in frozen
tissue biopsies at liquid nitrogen temperature [19]. This ensures the absence of artefacts
due to processing of tissues, such as homogenization, extraction etc.
EPR spectra were recorded at liquid nitrogen temperature (−196 ◦ C) with a Mag-
nettech MiniScope MS 200 EPR spectrometer (Magnettech Ltd., Berlin, Germany) at a
modulation frequency of 100 kHz and microwave frequency of 9.429 GHz. The settings
for NO-Hb spectra in blood samples were microwave power 8.3 mW and modulation
amplitude 5 G. NO-Hb complexes were recorded at 3300 ± 200 G and quantified by double-
integrating the EPR spectra. The settings for NO-Hb and Fe-NO complex in liver samples
were microwave power 30 mW and modulation amplitude 6 G. Liver spectra were recorded
at 3200 ± 500 G. The settings for p450 (g = 2.25), free radicals (g = 2.002), and succinate
dehydrogenase (g = 1.94) were microwave power 1 mW and modulation amplitude 5 G).
The intensity of the signals was recorded at 3200 ± 1000 G.

2.7. Analysis of Total Nitric Oxide

Total nitric oxide levels (NOx) were analyzed with Sievers 280i-NO Analyzer (General
Electric Company, Boston, MA, USA) as previously described [20]. Plasma samples were
injected through a septum into the glass vessel, where NO species were converted by a
redox active reagent (VCl3) to NO(g). Subsequent reaction with ozone causing photon
emission was detected as chemiluminescence intensity.

2.8. Sampling of Peritoneal and Bronchoalveolar Lavage

Bronchoalveolar lavage was collected as described elsewhere [21]. A 19-gauge needle
hub was inserted into the trachea and 3 mL of ice-cold phosphate-buffered saline (PBS) and
10% fetal bovine serum (FBS) were administered and aspirated slowly through the needle
hub. After transfer into a 15 mL tube (Greiner Bio-One, Kremsmünster, Austria), the fluid
was kept on ice. The procedure was repeated four times.
For collection of peritoneal cells, we used a protocol adapted from [22]. Briefly, the
peritoneum was exposed and 5 mL of ice-cold PBS with 3% FBS was injected into the
peritoneal cavity through a needle. A gentle massage to the abdomen was applied to
dislodge any attached cells into the PBS solution. The suspension was collected through
a 22-gauge needle into a syringe and transferred into a 15 mL tube, then kept on ice. The
above-described procedure was repeated five times.

2.9. Isolation of Cells

The suspension of bronchoalveolar and peritoneal cells was centrifuged at 400× g for
10 min at 4 ◦ C, the supernatant was discarded, and the cell number was counted with a Cell-
Dyn 3700 Hematology Analyzer (Abbott Laboratories, Lake Bluff, IL, USA). Cell counts in
blood and in peritoneal and bronchoalveolar fluids showed mainly polymorphonuclear
leukocytes (PMN, mainly neutrophil granulocytes; see Supplementary Figure S1). After the
blood count, blood samples were centrifuged at 300× g for 10 min at 4 ◦ C, then the plasma
and buffy coat were removed and the remaining blood pellet was incubated with a lysis
buffer containing 168 mM NH4Cl, 10 mM KHCO3, and 973 µM EDTA for 10 min at 4 ◦ C.
Cells were then washed twice with 10 ◦ C cold PBS and centrifuged at 400× g for 8 min
before finally performing the counts. For the in vitro mtAOX treatment, cell suspensions
were incubated at 37 ◦ C for 45 min with mitoTEMPO (500 nM) or vehicle (NaCl).

2.10. Ex Vivo ROS Measurement

The H2 O2 production in the cell suspension was assessed by N-acetyl-3,7-dihydroxy
phenoxazine (Amplex Red, Life Technologies, Eugene, OR, USA). Amplex Red is a sensitive
Biomolecules 2023, 13, 794 5 of 13

and chemically stable fluorogenic probe, and produces fluorescent resolufin with H2 O2
in a horseradish peroxidase (HRP)-catalyzed oxidation with excitation/emission maxima
at 563/587. The reaction stoichiometry of Amplex Red and H2 O2 is 1:1 [21]. Using a
fluorescence plate reader (POLARstar Omega 3MG, Labtech, Germany), white blood cells,
bronchoalveolar lavage, and peritoneal lavage cells (15 × 103 cells per well in Krebs buffer)
were incubated in black 96-well microplates (Greiner Cellstar) at 37 ◦ C with Amplex Red
(10 µM) and HRP (0.2 U mL−1 ). The fluorescence intensity was recorded for 30 min with
an excitation of 544 nm/emission of 590 nm and gain at 1200. The slope was calculated
from a 10 min interval between the 5th and 15th minute of the measurement.

2.11. Statistics
Statistical evaluation of the experimental results was performed using GraphPad
Prism(Version 9.4.1., GraphPad Software, Boston, MA, USA). The results are shown as
the mean ± standard error of the mean (SEM). Statistical significance was evaluated by
ANOVA followed by Post Hoc, Holm-Šídák’s multiple comparisons test unless indicated
otherwise in the figure legends; n numbers are indicated in the figure legends. Statistical
significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

3. Results
3.1. LPS Treatment Leads to Tissue Damage in an mtAOX-Dependent Way
As a first step, we validated our rat endotoxemia model (Figure 1).
By 16 h post-LPS challenge, an increase in aspartate aminotransferase AST (Figure 2a)
could be observed. MitoTEMPO pretreatment, however, could ameliorate this parenchymal
damage. According to the literature, an early peak of tumor necrosis factor (TNF)-α can be
seen at 2 h post-LPS in the plasma [2]. In this model, in vivo treatment with mitoTEMPO
neither influenced the release of early acute phase cytokines (TNF, IL-1) into the circulation
(Figure 2b,c) nor substantially decreased the levels of cytokines such as IL-4 and MCP-1
linked to the activation of monocytes (Figure 1d,e). In addition, we found that mitochon-
drial function in the liver is affected by LPS challenge (Figure 1f). As with the AST release,
addition of mitoTEMPO normalized the respiratory control ratio (Figure 1f).

3.2. In Vivo mitoTEMPO Treatment Does Not Influence Ex Vivo ROS Generation
Next, we examined the ex vivo ROS generation by immune cells isolated from periph-
eral blood, peritoneal fluid, and lavage from the bronchoalveolar system. However, we did
not observe any change in ROS generation in response to in vivo mitoTEMPO treatment
in any of the studied compartments (Figure 3a–c). Further, to test whether the capacity
of cells to generate ROS is affected by mitoTEMPO at all, we activated cells ex vivo by
phorbol 12-myristate 13-acetate (PMA); again, we did not find any difference in this case
(Figure 3d–f). Treatment with mitoTEMPO did not influence the relative counts of immune
cells in any of the three compartments (Supplementary Figure S1). These data suggest that
either ROS generation by immune cells is not regulated by mitoROS or mitoTEMPO did
not affect immune cells in vivo.

3.3. Ex Vivo mitoTEMPO Application Reduces ROS Production

To investigate the direct effects of mitoTEMPO on ROS production of immune cells,
cells were similarly extracted from all body compartments as described above. In this
case, mitoTEMPO was applied ex vivo. In contrast to the in vivo treatment, mitoTEMPO
substantially reduced the rate of ROS generation (Figure 4a–c). Interestingly, this inhibition
disappeared when cells were additionally activated ex vivo by PMA, with the exception of
bronchoalveolar lavage cells (Figure 4d–f).
 Biomolecules 2023, 13, x FOR PEER REVIEW 6 of 14
Biomolecules 2023, 13, 794 6 of 13

✱✱✱✱ ✱✱✱✱

1200 ✱✱✱✱

1000

800
LPS
600 LPS+M
400

200

0
0h 2h 4h 8h 16 h

(a) (b)

✱ ✱ ✱ ✱

✱✱✱✱ ✱✱✱✱ ✱ ✱

✱✱✱ ✱✱ ✱✱ ✱ ✱ ✱ ✱ ✱

(c) (d)

✱✱✱ ✱✱✱✱
complex I-linked
✱✱✱✱ ✱✱✱✱
40 ✱✱ ✱
control ratio [AU]

30
respiratory

20

10

0
ctrl LPS LPS+M

(e) (f)
Figure 2. Effect of mitochondria-targeted antioxidant (mitoTEMPO, M) on liver function and hu-
Figure 2. Effect of mitochondria-targeted antioxidant (mitoTEMPO, M) on liver function and humoral
moral immune response after LPS treatment: (a) time course of AST (b), TNF-alpha (c), IL1-alpha
immune response after LPS treatment: (a) time course of AST (b), TNF-alpha (c), IL1-alpha (d) IL-4 (e),
(d) IL-4 (e), and MCP1. The animals were treated with 106 U/kg (approx. 2 mg/kg) LPS or saline
and(control
MCP1. group)
The animals were treated
and observed for with
up to106
16 U/kg (approx.
h. Samples were2 mg/kg)
taken atLPS or saline
2/4/8/16 (control
h. (f) group)
Mitochondrial
andrespiratory
observed for up toratio.
control 16 h. Liver
Samples were taken
samples at 2/4/8/16
were taken at 16 h.h.The
(f) Mitochondrial
data are presentedrespiratory
as meancontrol
± SEM.
ratio. Liver samples
Statistical werewas
evaluation taken at 16 h. The
performed data are presented
by ANOVA followed as bymean ± SEM. Statistical
Holm-Šídák’s multiple evaluation
comparisons
wastest. n = 3–8. Statistical
performed by ANOVA significance
followed by is Holm-Šídák’s
indicated as follows:
multiple* pcomparisons
< 0.05; ** p <test.
0.01;n*** p < 0.001;
= 3–8. **** p <
Statistical
0.0001. is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
significance
 pacity of cells to generate ROS is affected by mitoTEMPO at all, we activated cells ex vivo
by phorbol 12-myristate 13-acetate (PMA); again, we did not find any difference in this
case (Figure 3d–f). Treatment with mitoTEMPO did not influence the relative counts of
immune cells in any of the three compartments (Supplementary Figure S1). These data
suggest that either ROS generation by immune cells is not regulated by mitoROS or mito-
Biomolecules 2023, 13, 794 7 of 13
TEMPO did not affect immune cells in vivo.

(a) (b) (c)

(d) (e) (f)

Figure
Figure 3. 3.
InIn vivo
vivo effects
effects of of
thethe mitochondria-targeted
mitochondria-targeted antioxidant
antioxidant mitoTEMPO
mitoTEMPO (M)ROS
(M) on on ROS genera-
generation
tion in blood and bronchoalveolar lavage after LPS treatment: blood (a,d) and peritoneal (b,e), and
in blood and bronchoalveolar lavage after LPS treatment: blood (a,d) and peritoneal (b,e), and
bronchoalveolar (c,f) lavage were collected under standardized conditions as described in the meth-
bronchoalveolar (c,f) lavage were collected under standardized conditions as described in the methods
ods section. ROS generation was measured in cells without activation with PMA (a–c) and after ex
section. ROS generation
vivo activation with PMA was measured
(d–f). in are
The data cellspresented
without activation
as mean ± with
SEM.PMA (a–c)
n = 5–7. and after
Statistical ex vivo
evaluation
activation with PMA (d–f). The data are presented as mean ± SEM. n
was performed by ANOVA followed by Holm-Šídák’s multiple comparisons test. = 5–7. Statistical evaluation
was performed by ANOVA followed by Holm-Šídák’s multiple comparisons test.
3.3. Ex Vivo mitoTEMPO Application Reduces ROS Production
3.4. EPR Reveals mitoTEMPO-Dependent Decrease in Free Radical and NO Signals
To investigate the direct effects of mitoTEMPO on ROS production of immune cells,
cellsInvestigation
were similarlyof extracted
redox-sensitive
from allintracellular paramagnetic
body compartments centersabove.
as described using InEPR
thistech-
case,
nique (Figure 5a) revealed that their redox state is responsive to both LPS and
mitoTEMPO was applied ex vivo. In contrast to the in vivo treatment, mitoTEMPO sub- mitoTEMPO.
Both substances
stantially increased
reduced the ratetheofintensity of the signal
ROS generation coming
(Figure 4a–c).from p450 (Figure
Interestingly, this5b), while
inhibition
the free radical signal, which originates predominantly from mitochondrial Q10, was in-
disappeared when cells were additionally activated ex vivo by PMA, with the exception of
creased by LPS (Figure 5c), though this increase was diminished by mitoTEMPO (Figure 5c).
bronchoalveolar lavage cells (Figure 4d–f).
The signal g = 1.94, which is usually attributed to succinate dehydrogenase of mitochon-
dria, was increased by LPS but did not change in response to mitoTEMPO (Figure 5d).
In LPS-treated rats, the concentration of mononitrosyl-hemoglobin-complexes (NO-Hb)
determined in liver tissue (Figure 5f) was higher than in the blood (Figure 5e), support-
ing the assumption that liver is a (the) prominent NO source, eventually higher than the
vasculature and the blood itself. Increased concentrations of NO-Hb were attenuated by mi-
toTEMPO (Figure 5e,f). Similar changes were observed with liver dinitrosyl iron complexes
(Fe-NO) reporting intracellular NO levels (Figure 5g). In addition, NOx levels in plasma
after LPS treatment, determined by ozone-chemiluminescence technology, were increased
(Figure 5h). This increase could be reduced by mitoTEMPO treatment (Figure 5h).
 Biomolecules 2023, 13, x FOR PEER REVIEW 8 of 14
Biomolecules 2023, 13, 794 8 of 13

✱
✱✱
✱

(a) (b) (c)

(d) (e) (f)

Figure 4. Ex vivo effects of mitochondria-targeted antioxidant mitoTEMPO (M) on ROS generation
Figure 4. Ex vivo effects of mitochondria-targeted antioxidant mitoTEMPO (M) on ROS generation
in blood and peritoneal and bronchoalveolar lavage exposed to LPS. Blood (a,d) and peritoneal (b,e),
in blood and peritoneal and bronchoalveolar lavage exposed to LPS. Blood (a,d) and peritoneal
and bronchoalveolar (c,f) lavage were collected under standardized conditions as described in the
(b,e), and bronchoalveolar
methods (c,f) lavage
section. ROS generation was were collected
measured under
in cells standardized
without activationconditions
with PMA as described
(a–c) and after
inexthe methods section. ROS generation was measured in cells without activation with PMA
vivo activation with PMA (d–f). The data are presented as mean ± SEM. n = 2–6. Statistical evalu- (a–c)
and after ex vivo activation with PMA (d–f). The data are presented as mean
ation was performed by ANOVA followed by Holm-Šídák’s multiple comparisons test. Statistical ± SEM. n = 2–6.
Biomolecules 2023, 13, x FOR PEER REVIEW
Statistical evaluation
significance was performed
is indicated as follows: *by
p <ANOVA
0.05; ** pfollowed 9
< 0.01. by Holm-Šídák’s multiple comparisons of 14

test. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01.

3.4. EPR Reveals mitoTEMPO-Dependent Decrease in Free Radical and NO Signals
Investigation of redox-sensitive intracellular paramagnetic centers using EPR tech-
nique (Figure 5a) revealed that their redox state is responsive to both LPS and mito-
✱✱✱✱
TEMPO. Both substances increased the intensity of the signal coming from p450 (Figure
✱✱ ✱✱

5b), while the free radical signal, which originates predominantly from mitochondrial
Q10, was increased by LPS (Figure 5c), though this increase was diminished by mito-
TEMPO (Figure 5c). The signal g = 1.94, which is usually attributed to succinate dehydro-
genase of mitochondria, was increased by LPS but did not change in response to mito-
TEMPO (Figure 5d). In LPS-treated rats, the concentration of mononitrosyl-hemoglobin-
complexes (NO-Hb) determined in liver tissue (Figure 5f) was higher than in the blood
(Figure 5e), supporting the assumption that liver is a (the) prominent NO source, eventu-
ally higher than the vasculature and the blood itself. Increased concentrations of NO-Hb
were attenuated by mitoTEMPO (Figure 5e,f). Similar changes were observed with liver
dinitrosyl iron complexes (Fe-NO) reporting intracellular NO levels (Figure 5g). In addi-
(a) (b)
tion, NOx levels in plasma after LPS treatment, determined by ozone-chemiluminescence
technology,
Figure 5. Cont. were increased (Figure 5h). This increase could be reduced by mitoTEMPO
✱

treatment (Figure 5h). ✱✱✱ ✱

✱✱✱✱ ✱

✱
✱✱✱✱ ✱

(c) (d) (e)

Biomolecules 2023, 13, 794 9 of 13

(a) (b)

✱✱✱ ✱

✱✱✱✱ ✱

✱
✱✱✱✱ ✱

(c) (d) (e)

✱
✱
✱✱✱
2500 ✱✱✱✱
** ✱

2000

1500

1000

500

0
ctrl LPS LPS+M

(f) (g) (h)

Figure
Figure 5. 5.Redox
Redoxstate
stateofofparamagnetic
paramagneticcenters:
centers: EPR
EPR spectrum
spectrum of of liver
liver tissue
tissue (a);
(a);changes
changesininthe
thein-
tensity of the cyt P450 signal (g = 2.25) (b); changes in the intensity of tissue free radical signal (g =
intensity of the cyt P450 signal (g = 2.25) (b); changes in the intensity of tissue free radical signal
2.002) (c); changes in the intensity of succinate dehydrogenase signal (g = 1.94) (d); NO-Hb levels in
(g = 2.002) (c); changes in the intensity of succinate dehydrogenase signal (g = 1.94) (d); NO-Hb levels
circulating blood (e) and blood in liver vessels (f); Fe-NO levels in liver cells (g); NOx level in plasma
in (h).
circulating
The data blood (e) and blood
are presented as meanin liver vessels
± SEM. (f); Fe-NO
Statistical levels in
evaluation liver
was cells (g); by
performed NOx level in
ANOVA fol-
plasma (h). The data are presented as mean ± SEM. Statistical evaluation was performed
lowed by Holm-Šídák’s multiple comparisons test. n = 4–10. Statistical significance is indicated as by ANOVA
followed
follows:by* pHolm-Šídák’s multiple
< 0.05; ** p < 0.01; *** pcomparisons
< 0.001; **** ptest. n = 4–10. Statistical significance is indicated as
< 0.0001.
follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
4. Discussion
4. Discussion
In the present study, we employed a rodent model, which is extensively described in
In the present study, we employed a rodent model, which is extensively described
the literature, to investigate systemic inflammation and consequent organ damage. Bacte-
in the literature, to investigate systemic inflammation and consequent organ damage.
rial endotoxin (LPS) treatment is known to lead to a dose-dependent increase in mortality
Bacterial endotoxin (LPS) treatment is known to lead to a dose-dependent increase in
along with elevation of organ damage markers and nitric oxide levels, as well as substan-
mortality along with elevation of organ damage markers and nitric oxide levels, as well
tial changes in plasma cytokine pattern [23,24]. It is generally accepted that an inflamma-
as substantial changes in plasma cytokine pattern [23,24]. It is generally accepted that an
tory activation of cytokines orchestrated by and acting through ROS play a central role in
inflammatory activation of cytokines orchestrated by and acting through ROS play a central
end-organ damage [25].
role in end-organ damage [25].
Mitochondria-targeted antioxidants such as mitoTEMPO are widely employed to
elucidate functional aspects of ROS-dependent mechanisms. Because we have already
investigated the effect of mitoTEMPO on control tissues in previous studies [2,26], we did
not include this group here. Consequently, the conclusions are limited to comparisons
between control and LPS on one hand, and on the other LPS and LPS with mitoTEMPO.
In the present study, mitoTEMPO treatment successfully mitigated the tissue damage
to the liver parenchyma, as evidenced by AST levels. This is in line with previous publi-
cations [2]. We observed that the respiratory control ratio is increased in response to LPS
and normalized upon addition of mitoTEMPO. It has already been reported that upon
inflammatory conditions the mitochondrial respiratory function can respond by a decrease
or an increase in the capacity of the electron transport chain [27]. In our previous studies on
rodents, we observed an increase in the State 3 respiration [28], as we show here, while in
experiments with peritonitis in pigs the respiratory activity was decreased [29]. The reason
that mitochondrial function is upregulated in certain cases and downregulated in others is
not completely clear. However, the normalization of mitochondrial function to its control
 inflammatory conditions the mitochondrial respiratory function can respond by a de-
crease or an increase in the capacity of the electron transport chain [27]. In our previous
studies on rodents, we observed an increase in the State 3 respiration [28], as we show
here, while in experiments with peritonitis in pigs the respiratory activity was decreased
Biomolecules 2023, 13, 794 [29]. The reason that mitochondrial function is upregulated in certain cases and downreg- 10 of 13
ulated in others is not completely clear. However, the normalization of mitochondrial
function to its control values by mitoTEMPO suggests that the changes in mitochondrial
function observed here reflect pathological changes in the liver and that these changes are
values by mitoTEMPO suggests that the changes in mitochondrial function observed here
mediated by mitoROS.
reflect pathological changes in the liver and that these changes are mediated by mitoROS.
According to the literature, an early peak of TNF-α can be seen at 2 h post-LPS [2].
According to the literature, an early peak of TNF-α can be seen at 2 h post-LPS [2].
TNF-α and IL-1 are central cytokines in the acute systemic inflammatory response, on the
TNF-α and IL-1 are central cytokines in the acute systemic inflammatory response, on the
one hand exerting direct effects on hepatocytes, inducing apoptosis and necrosis [30], and
one hand exerting direct effects on hepatocytes, inducing apoptosis and necrosis [30], and
on the other hand sending later cytokines into action. Our data suggest that fast (2 h and
on the other hand sending later cytokines into action. Our data suggest that fast (2 h and
earlier) release of TNF/IL-1 at the beginning of acute inflammatory response orchestrates
earlier) release of TNF/IL-1 at the beginning of acute inflammatory response orchestrates
the immune response rather than directly contributing to the liver damage. This may in-
the immune response rather than directly contributing to the liver damage. This may
deed be possible, as the first wave of TNF/IL-1 is released from membrane-bound pools
indeed be possible, as the first wave of TNF/IL-1 is released from membrane-bound pools
of
of these inflammatory mediators
these inflammatory mediators and
and not
not from
from aa pool which needs
pool which needs to
to be
be synthetized
synthetized de de
novo. This does not preclude the fact that in the later second wave of their release,
novo. This does not preclude the fact that in the later second wave of their release, when when
they
they are upregulated on
are upregulated on the
the genetic
genetic level,
level, their
their levels
levels will
will be
be sensitive
sensitive to
to mitoTEMPO
mitoTEMPO
(Figure 6).
(Figure 6).

Figure 6.
Figure Schematic presentation
6. Schematic presentation of
of the
the time
time course
course of
of early
early cytokine
cytokine response
response and
and hepatocellular
hepatocellular
damage after intravenous LPS challenge with and without mtAOX treatment in rats.
damage after intravenous LPS challenge with and without mtAOX treatment in rats. LPS-induced
LPS-induced
hepatocellular damage, reflected by serum AST levels, is shown on the same timescale. AST can be
seen rising slightly
slightly with
withthe
thetime
timefollowing
followingLPS LPSinjection,
injection,reaching
reaching significance
significance compared
compared to base-
to baseline
line levels
levels at 8 at 8 h post-LPS.
h post-LPS. MtAOX
MtAOX treatment
treatment shows
shows a strong
a strong reduction
reduction in circulating
in circulating AST
AST levels
levels 1616
h
h post-LPS, indicating a protective effect of mtAOX, and conversely a key role of
post-LPS, indicating a protective effect of mtAOX, and conversely a key role of mtROS in organ mtROS in organ
damage
damage induced
induced by endotoxemia.
by endotoxemia.

Assuming effective
effective intramitochondrial radical scavenging properties of mtAOX, we
can conclude the following based on the experimental data. Beneficial
Beneficial effects
effects of mtAOX
on hepatocellular
hepatocellular injury
injury by 16 h are not directly linked to TNF-α, as mtAOX prevented
AST release despite no alterations in TNF levels. Either later cytokines in the cascade are
ROS-sensitive, or another process is contributing to cellular damage.
To determine whether ROS production in different body compartments can be modu-
lated by mitoTEMPO, we examined ROS generation by immune cells in the blood, peri-
toneal fluid, and lavage from the bronchoalveolar system. We predominantly obtained
PMNs from the three body compartments. Considering the central role of circulating PMNs
in this systemic inflammation model, which is not TNF-α-dependent, an explanation for
the protective effects could be a direct effect of mtAOX on PMNs. Although these cells
have only a few functional mitochondria, a growing body of evidence suggests important
functions of this organelle in initiation and migration [31], and Dikalov [32] and Daiber [33]
have previously reported a feed-forward loop between mtROS and nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase activity. Theoretically, by inhibiting mtROS less
ROS is formed by NADPH oxidase, resulting in less parenchymal cellular damage. Thus,
we designed an experiment to investigate a direct effect of mtAOX on PMNs. There was
no difference between PMA-induced ROS production of treated and non-treated groups,
implying no causative role of reduced oxidative bursts of circulating immune cells capable
of eliciting oxidative damage in the target organ in this setting.
We failed to reproducibly determine ROS generation of controls, and analyzed only
the cells obtained from animals treated with LPS. In contrast to our expectations, in vivo
injection of mitoTEMPO did not influence the rate of ROS generation in any of the examined
Biomolecules 2023, 13, 794 11 of 13

compartments. Interestingly, the ex vivo treatment of the same cells with mitoTEMPO
substantially reduced ROS generation. We assume that mitoTEMPO is distributed inside the
body within the tissues; however, at the time when PMNs generate ROS it is possible that
mitoTEMPO may not be present in sufficiently high concentrations in the corresponding
compartments. In addition, it is possible that these cells might not be completely activated,
as external treatment with PMA induced an additional release of ROS. The ROS release
induced by PMA was not sensitive to mitoTEMPO. These data suggest that effect of
mitoTEMPO in terms of ROS release is not mediated by activated immune cells.
Our observation that mitoTEMPO attenuated ROS generation ex vivo when exposure
occurred prior to PMA treatment suggests that the generation of extracellular ROS in all
three compartments upon treatment with LPS is controlled by mitoROS. This may not
be the case when cells are activated by PMA. Indeed, PMA may activate immune cells
in a mitoROS-independent manner by directly activating protein kinase C (PKC), and
subsequently NADPH oxidase. However, LPS is known to act via toll-like receptors (TLR),
which trigger the activation of NADPH-oxidases over mitochondrial ROS [34].
When analyzing the EPR spectra of the liver we observed specific changes of redox
sensitive paramagnetic centers, particularly those involved in mitochondrial ROS genera-
tion and scavenging. We observed an increase in the Q10 signal in response to LPS, which
was attenuated by mitoTEMPO. Q10 is an antioxidant in its reduced state, and it is able
to scavenge ROS to yield stable free radicals. The latter can be detected by EPR. In vivo
treatment with mitoTEMPO reduced this signal to its normal levels. Induction of iNOS is a
sign of ongoing inflammation, and it has previously been shown that iNOS is upregulated
in our model [2]. In our experiments, we observed increased NO levels in liver tissue and
blood. The concentration of NO in liver tissue was higher than in the blood, suggesting that
NO is formed predominantly in the liver (and probably other tissues) and then released
into blood. In vivo treatment with mitoTEMPO reduced the NO levels elevated by LPS.
Based on the data presented here, and according to the literature [9], it can be con-
cluded that classical early pro-inflammatory cytokines (TNF-α, IL-6) alone are not direct
mediators of tissue damage in vivo. MtAOX treatment prevented hepatocellular damage
despite obvious elevation of TNF-α in the circulation. Our data suggest that inflammatory
mediators orchestrate systemic immune response, rather than directly contributing to
ROS-mediated liver damage. Because ROS can originate in extracellular and intracellu-
lar compartments, it is expected that the mitochondrial antioxidant mitoTEMPO should
prevent ROS-induced organ damage. This is supported by the fact that mitochondrial
ROS regulate the release of intracellular and extracellular ROS from other sources [3].
Consequently, mitoTEMPO influences the redox status of liver cells, which can contribute
to its local beneficial effect, though it may not be sufficient to reduce the systemic ROS
generation by immune cells. The latter is likely due to the major portion of mitoTEMPO
being absorbed by tissues and only a small portion being absorbed by immune cells. This
may be explained by the volume/absorption surface of the tissues, which is much higher
than the surface of immune cells.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/biom13050794/s1, Supplementary Figure S1: Effect of mitoTEMPO
on cell counts in blood and peritoneal and bronchoalveolar fluids.
Author Contributions: Conceptualization, A.V.K.; methodology, A.T.M. and S.D.; validation, A.T.M.,
A.W., S.D. and A.V.K.; formal analysis, A.T.M., A.W. and S.D.; investigation, A.T.M. and S.D.; re-
sources, A.V.K.; data curation, A.W.; writing—original draft preparation, A.W. and A.V.K.;
writing—review and editing, A.T.M., A.W., S.D. and A.V.K.; supervision, A.V.K.; project admin-
istration, A.W. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: All interventions were conducted in compliance with the
Guide for the Care and Use of Laboratory Animals published by the National Institute of Health with
approval from the Animal Protocol Review Board (M58003956-2011-9, no. 815758/2014/16).
Biomolecules 2023, 13, 794 12 of 13

Informed Consent Statement: Not applicable.

Data Availability Statement: The data of this study are available on request from the corresponding
author, [A.V.K.].
Conflicts of Interest: The authors declare no conflict of interest.

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