Ci a ion: Elexpe, A.; Sánchez-
Sánchez, L.; Tolen ino-Co ez, T.;
As iga aga, E.; To ecilla, M.;
Ba eda-Gómez, G. Analysis o
Mi ochond ial Func ion in Cell
Memb anes as Indica o o Tissue
Vulne abili y o D ugs in Humans.
Biomedicines 2022,10, 980.
h ps://doi.o g/10.3390/
biomedicines10050980
Academic Edi o s: Sil ia O ega-
Gu ie ez, Ami a a Saei Diba a
and Ma ía L. López-Rod íguez
Recei ed: 31 Ma ch 2022
Accep ed: 21 Ap il 2022
Published: 23 Ap il 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
biomedicines
A icle
Analysis o Mi ochond ial Func ion in Cell Memb anes as
Indica o o Tissue Vulne abili y o D ugs in Humans
Ane Elexpe 1,2, Lau a Sánchez-Sánchez 1,3 , Ta son Tolen ino-Co ez 1, Egoi z As iga aga 1, Ma ía To ecilla 2
and Gab iel Ba eda-Gómez 1,*
1Resea ch and De elopmen Depa men , IMG Pha ma Bio ech S.L, 48160 De io, Spain;
[email p o ec ed] (A.E.); [email p o ec ed] (L.S.-S.); [email p o ec ed] (T.T.-C.);
[email p o ec ed] (E.A.)
2Depa men o Pha macology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y
UPV/EHU, 48940 Leioa, Spain; [email p o ec ed]
3Ins i u e o Molecula Gene ics and Biology (IBGM), Uni e si y o Valladolid-CSIC, 47003 Valladolid, Spain
*Co espondence: gab iel.ba [email p o ec ed]; Tel.: +34-94-4316-577; Fax: +34-94-6013-455
Abs ac :
D ug side e ec s a e one o he main easons o ea men wi hd awal du ing clinical
ials. Reac i e oxygen species o ma ion is in ol ed in many o he d ug side e ec s, mainly by
in e ac ing wi h he componen s o he cellula espi a ion. Thus, he ea ly de ec ion o hese e ec s
in he d ug disco e y p ocess is a key aspec o he op imiza ion o pha macological esea ch. To his
end, he supe oxide o ma ion o a se ies o d ugs and compounds wi h an idep essan , an ipsycho ic,
an icholine gic, na co ic, and analgesic p ope ies was e alua ed in isola ed bo ine hea memb anes
and on cell memb ane mic oa ays om a collec ion o human issues, oge he wi h speci ic inhibi o s
o he mi ochond ial elec on anspo chain. Fluphenazine and PB28 p omo ed simila e ec s o
hose o o enone, bu wi h lowe po ency, indica ing a di ec ac ion on mi ochond ial complex I.
Mo eo e , ne azodone, a d ug wi hd awn om he ma ke due o i s mi ochond ial hepa o oxic
e ec s, e oked he highes supe oxide o ma ion in human li e cell memb anes, sugges ing he
po en ial o his echnology o an icipa e ad e se e ec s in p eclinical phases.
Keywo ds: mic oa ay; supe oxide; an ipsycho ic; mi ochond ia
1. In oduc ion
Reac i e oxygen species (ROS) o ma ion is a physiological p ocess p oduced by di e -
en pa hways and con olled by di e se an ioxidan mechanisms; howe e , i can u n in o
a pa hological s a e due o an imbalance be ween oxidan and an ioxidan compounds [
1
].
These compounds a e a se ies o adical and non adical oxygen species o med upon
incomple e oxygen educ ion [
2
] whose augmen a ion can induce lipid pe oxida ion and
mul iple al e a ions in p o eins and nuclei acids. They can be p oduced by se e al sou ces,
such as NADPH oxidase (NOX) enzyme amily [
3
], dual oxidase (DUOX) [
4
], monoamine
oxidase (MAO) [
5
], pe oxisomes, and mi ochond ial elec on anspo chain (mETC) [
6
].
The di e en ypes o NADPH oxidases a e implica ed in eac i e oxygen species o ma ion
in a a ie y o issues, such as b ain, hea , o li e [
7
]; speci ically, NOX2, NOX3, and
NOX4 a e exp essed h oughou he ne ous sys em [
7
], whe eas o he iso o ms such as
NOX5 a e ound only in he lymph nodes and spleen [
8
]. Addi ionally, dual oxidase en-
zymes (DUOX) p oduce hyd ogen pe oxide di ec ly o indi ec ly [
7
]. Monoamine oxidase
is an enzyme loca ed in he mi ochond ial ou e memb ane, wi h wo di e en iso o ms
(MAO-A and MAO-B). One o i s ca aly ic p oduc s is hyd ogen pe oxide [
9
,
10
] whose
accumula ion p o okes he damage o many cell ypes including neu al cells [
11
] and is
implica ed in many b ain pa hologies [
12
], speci ically Alzheime ’s [
13
] and Pa kinson’s
disease [
14
], F ied eich A axia [
15
], mul iple scle osis [
16
], and some psycho ic diso de s
such as bipola diso de [
17
] and schizoph enia [
18
,
19
]. Among hese p o eins, he main
Biomedicines 2022,10, 980. h ps://doi.o g/10.3390/biomedicines10050980 h ps://www.mdpi.com/jou nal/biomedicines
Biomedicines 2022,10, 980 2 o 16
ones esponsible o cy osolic hyd ogen pe oxide and o he ROS a e hose in ol ed in he
mi ochond ial elec on anspo chain and cy och ome P450 enzyma ic sys em [
20
]. On
he one hand, he oxida i e phospho yla ion (OXPHOS) p ocess, pe o med in he mi o-
chond ial inne memb ane and composed o i e enzyma ic complexes, is he main sou ce
o ene gy as well as eac i e oxygen species. Mi ochond ia a e o ganelles p esen in mos
euka yo ic cells ha can pe o m a a ie y o me abolic unc ions [
21
], such as oxida i e
phospho yla ion [
22
,
23
] and me aboli e egula ion. They a e also implica ed in homeos a ic
signaling [
22
] and lipid biosyn he ic pa hways [
23
]. P incipally, supe oxide is p oduced by
mi ochond ial complexes I and III [
24
]. Thei dys egula ion leads o an ATP p oduc ion
dec ease, an inc ease in oxida i e s ess, and may e en ini ia e apop o ic p ocesses leading
o d ug side e ec s [
25
]. On he o he hand, cy och ome P450 enzymes a e in ol ed in he
me aboliza ion o compounds [
20
]. These enzymes oxygena e o ganic xenobio ic subs a es
and ca alyze he educ ion o molecula oxygen simul aneously; i i is no pe o med
co ec ly, oxygen uncouples om he subs a e and leads o ROS o ma ion [26].
In physiological s a es, enzyma ic and nonenzyma ic an ioxidan mechanisms egu-
la e he cellula edox s a us by con olling he p oduc ion o second messenge s [
27
,
28
]
and ansc ip ion ac o s [
29
] in di e se signaling pa hways. Fo ins ance, supe oxide
dismu ase (SOD) sca enges supe oxide adicals [
29
], while ca alase [
30
], glu a hione pe -
oxidase, and pe oxy edoxin [
24
] p o ec he cell om he ad e se e ec s o hyd ogen
pe oxide h ough i s b eakdown in o wa e and oxygen. Ne e heless, he exposu e o
some s ess condi ions, such as di e en pa hologies o e en medica ion in ake, can make
hese mechanisms insu icien .
In a ce ain way, many d ugs ha a e being es ed in clinical ials a e wi hd awn in
p eclinical phases because o hei high oxici y due o he o ma ion o eac i e oxygen
species. Conside ing ha he majo sou ce o hese small molecules is he mi ochond ial
elec on anspo chain, hei dys unc ion can gene a e an ene ge ic educ ion ha can ha e
a pa icula ly p onounced impac on he e iciency o neu onal unc ions compa ed o o he
issues [
31
]. Schizoph enia is a cogni i e diso de ha a ec s 1% o he popula ion in which
dissocia ion o hough dis up ion is expe ienced [
32
]. This illness deno es al e a ions
in he neu o ansmission o dopamine inside he mesolimbic sys em and mesoco ical
pa hway [
33
]. Mo eo e , i s -episode schizoph enic pa ien s ha e dec eased SOD ac i i y
making hem p one o su e om oxida i e s ess condi ions [
34
]. I has been obse ed
ha some an ipsycho ics ha e been associa ed wi h mETC inhibi ion causing an inc ease in
eac i e oxygen species o ma ion [
35
] by complex I inhibi ion [
36
]. Complex I inhibi ion
con ibu es o a u he educ ion o mi ochond ial ene gy p oduc ion, which is al eady
limi ed in some cogni i e diso de s, such as bipola diso de [
31
] and schizoph enia [
31
,
37
].
Fo ins ance, clozapine can induce oxida i e s ess and apop osis in neu ophil cells [
38
],
whe eas ano he an ipsycho ic d ugs, such as pen azocine [
39
], seem o educe i . In
addi ion, hese oxida i e dysbalanced condi ions ha e been ela ed o ex apy amidal
ad e se e ec s [40,41].
Oxida i e s ess is also a con ibu ing ac o in o he men al diso de s, such as de-
p ession [
42
], he mos common o his ype o disease. I is cha ac e ized by apa hy,
anhedonia, sleep dis u bance, and psychomo o e a da ion [
43
]. Among he an idep es-
san d ugs, ne azodone, an an agonis o he 5-hyd oxi ip amine (5-HT) ecep o , was
used o se e al yea s o ea dep ession un il i was wi hd awal om he ma ke . I was
epo ed ha ne azodone caused li e oxici y and hepa ic ailu e [
44
] due o he inhibi ion
o cy och ome P450 [
45
], in e e ence wi h OXPHOS enzymes, and gene a ion o eac i e
oxygen species [
44
]. O he d ugs, such as clozapine o luphenazine, ha e also been asso-
cia ed wi h some hepa ic al e a ions [
46
] and ca diac al e a ions such as ca diomyopa hy
o myoca di is [47].
Alzheime ’s disease, he mos common neu odegene a i e cause o demen ia [
48
],
is ano he men al illness in which oxida i e s ess seems o play a pa icula ole
[48,49]
.
This neu ogene a i e diso de has al e a ions such as he deposi o amyloid-be a plaques,
dys egula ion o cen al ne ous sys em immune esponse [
50
], dys unc ion o oxida i e
Biomedicines 2022,10, 980 3 o 16
phospho yla ion, and eac i e oxygen species gene a ion due o mi ochond ial dis unc-
ion [
51
]. Rega ding he pa hologic e ec s o Alzheime ’s disease, ligands o sigma 1
and 2 ecep o s can egula e he CNS immune esponse and modula e amyloid-be a p o-
duc ion [
35
,
50
]. Fu he mo e, i has been epo ed ha sigma ecep o ligands s ongly
inc eased mi ochond ial supe oxide adicals [52].
As hese medicines a e usually es ed in samples om animal models
in i o
and
in i o
, hei side e ec s could be di e en o hose p esen in human samples. In his
ega d, di e en luo escence spec oscopy and colo ime ic echniques [
53
] ha e been
ca ied ou o de e mine he p oduc ion o ROS in a a ie y o o ganisms. In his sense, he
aim o his s udy was o analyze he supe oxide o ma ion e oked by d ugs and compounds
wi h an ipsycho ic, an icholine gic, na co ic, and analgesic p ope ies in isola ed bo ine
hea memb anes and on human cell memb ane mic oa ays (CMMAs). These CMMAs
(Figu e 1) consis ed o a collec ion o memb anes isola ed om 10 human issues, which
main ain he memb ane en i onmen and p o ein unc ionali y, enabling hei use in
supe oxide assays [54].
Biomedicines 2022, 10, x FOR PEER REVIEW 3 o 18
This neu ogene a i e diso de has al e a ions such as he deposi o amyloid-be a plaques,
dys egula ion o cen al ne ous sys em immune esponse [50], dys unc ion o oxida i e
phospho yla ion, and eac i e oxygen species gene a ion due o mi ochond ial dis unc-
ion [51]. Rega ding he pa hologic e ec s o Alzheime ’s disease, ligands o sigma 1 and
2 ecep o s can egula e he CNS immune esponse and modula e amyloid-be a p oduc-
ion [35,50]. Fu he mo e, i has been epo ed ha sigma ecep o ligands s ongly in-
c eased mi ochond ial supe oxide adicals [52].
As hese medicines a e usually es ed in samples om animal models in i o and in
i o, hei side e ec s could be di e en o hose p esen in human samples. In his ega d,
di e en luo escence spec oscopy and colo ime ic echniques [53] ha e been ca ied ou
o de e mine he p oduc ion o ROS in a a ie y o o ganisms. In his sense, he aim o his
s udy was o analyze he supe oxide o ma ion e oked by d ugs and compounds wi h
an ipsycho ic, an icholine gic, na co ic, and analgesic p ope ies in isola ed bo ine hea
memb anes and on human cell memb ane mic oa ays (CMMAs). These CMMAs (Figu e
1) consis ed o a collec ion o memb anes isola ed om 10 human issues, which main ain
he memb ane en i onmen and p o ein unc ionali y, enabling hei use in supe oxide
assays [54].
Figu e 1. Scheme o he me hodology used o e alua e he issue-speci ic d ug-media ed supe oxide
o ma ion in human issues. CMMAs we e composed o human cell memb anes om 10 di e en
o gans and issues. D ugs and compounds wi h an ipsycho ic, an idep essan , an icholine gic, na -
co ic, and analgesic p ope ies we e incuba ed wi h CMMAs, and supe oxide o ma ion was de-
ec ed by a colo ime ic assay. CMMAs we e digi alized, and he da a a e no maliza ion we e an-
alyzed o de e mine ROS o ma ion in each human issue.
2. Ma e ials and Me hods
2.1. D ugs and Reagen s
Ni o e azolium blue chlo ide (NBT), 3,3′-Diaminobenzidine (DAB), be a-nico ina-
mide adenine dinucleo ide (NADH), sodium succina e dibasic (SDH), decylubiquinone
(dUQ), o enone, an imycin A, sodium azide, cy och ome c om equine hea , olanzapine,
and luphenazine dichlo ide we e pu chased om Sigma-Ald ich (Sain Louis, MO,
USA). (−)-Pen azocine, PPCC oxala e, PB 28 dihyd ochlo ide, N’N dime hyl yp amine
(DMT), BD 1047 dihyd ob omide, ne azodone hyd ochlo ide, dex ome ho phan hyd o-
chlo ide, and NE 100 hyd ochlo ide we e pu chased om Toc is Bioscience (B is ol, UK).
Figu e 1.
Scheme o he me hodology used o e alua e he issue-speci ic d ug-media ed supe oxide
o ma ion in human issues. CMMAs we e composed o human cell memb anes om 10 di e en
o gans and issues. D ugs and compounds wi h an ipsycho ic, an idep essan , an icholine gic,
na co ic, and analgesic p ope ies we e incuba ed wi h CMMAs, and supe oxide o ma ion was
de ec ed by a colo ime ic assay. CMMAs we e digi alized, and he da a a e no maliza ion we e
analyzed o de e mine ROS o ma ion in each human issue.
2. Ma e ials and Me hods
2.1. D ugs and Reagen s
Ni o e azolium blue chlo ide (NBT), 3,3
0
-Diaminobenzidine (DAB), be a-nico inamide
adenine dinucleo ide (NADH), sodium succina e dibasic (SDH), decylubiquinone (dUQ),
o enone, an imycin A, sodium azide, cy och ome c om equine hea , olanzapine, and
luphenazine dichlo ide we e pu chased om Sigma-Ald ich (Sain Louis, MO, USA).
(
−
)-Pen azocine, PPCC oxala e, PB 28 dihyd ochlo ide, N’N dime hyl yp amine (DMT),
BD 1047 dihyd ob omide, ne azodone hyd ochlo ide, dex ome ho phan hyd ochlo ide,
and NE 100 hyd ochlo ide we e pu chased om Toc is Bioscience (B is ol, UK).
2.2. Tissue Samples
Hea samples om Bos au us we e supplied by Llodio municipal slaugh e house (Ala a,
Spain). Human biopsy issues we e supplied by he AMSBIO (Abingdon,
Ox o dshi e, UK
)
issue bank acco ding o i s e hical p o ocols (Table S2).
Biomedicines 2022,10, 980 4 o 16
2.3. Cell Memb ane Mic oa ay Fab ica ion
Cell memb ane mic oa ays we e composed o a collec ion o cell memb ane ho-
mogena es isola ed om di e en human issues (li e , jejunum, lung, enal medulla, enal
co ex, ad enal gland, myoca dium, adipose issue, duodenum, and spleen). B ie ly, sam-
ples we e homogenized using a dispe se (Ul a-Tu ax
®
T10 basic, IKA, S au en, Ge many)
o a Te lon-glass g inde (Heidolph RZR 2020, Schwabach, Ge many) in 20 olumes o
homogeniza ion bu e (1 mM EGTA, 3 mM MgCl
2
, and 50 mM T is-HCl, pH 7.4) sup-
plemen ed wi h 250 mM suc ose. The c ude homogena e was subjec ed o a 1500- pm
cen i uga ion (Alleg aTM X 22R cen i uge, Beckman Coul e , B ea, CA, USA) o 5 min a
4
◦
C, and he esul an supe na an was collec ed and cen i uged a 18,000 g (Mic o uge
®
22R cen i uge, Beckman Coul e , B ea, CA, USA) o 15 min (4
◦
C). The pelle was washed
in 20 olumes o homogenized bu e and ecen i uged unde he same condi ions. The
ubes we e inally decan ed, and he pelle s we e ozen a −80 ◦C, excep o one aliquo ,
which was used o de e mine he p o ein concen a ion. P o ein concen a ion was de e -
mined by he B ad o d me hod [55,56] and adjus ed o he inal concen a ion.
Memb ane homogena es we e esuspended in bu e and p in ed on o glass slides
using a noncon ac mic oa aye (Nanoplo e NP 2.1), placing 3 eplica es o each sam-
ple (7 nL/spo ) on o p eac i a ed glass mic oscope slides. Memb ane homogena es o
each issue we e ob ained om h ee di e en indi iduals. The p in ing was ca ied ou
unde con olled humidi y ( ela i e humidi y 60%) a a con olled empe a u e o 4
◦
C.
CMMAs we e s o ed a
−
20
◦
C un il usage [
54
]. CMMAs we e alida ed be o e usage by
di e en me hods including B ad o d s aining o p o ein de e mina ion, enzyme ac i i y
assays (NADH oxido educ ase, succina e dehyd ogenase, and cy och ome c oxidase), and
adioligand binding assays. [54,57–61]
2.4. De e mina ion o D ug E ec s on Supe oxide Fo ma ion P omo ed by NADH in Isola ed
Bo ine Hea Cell Memb anes
The NADH-ubiquinone oxido educ ase ac i i y assay was pe o med on cell mem-
b anes isola ed om bo ine hea issue. Fo his pu pose, memb ane homogena es
(0.1 mg/mL) we e incuba ed in he p esence o 0.35 mM NADH and 0.5 mg/mL NBT in
phospha e bu e (5 mM; pH 7.4) o 3 h a 25
◦
C wi h inc easing concen a ions ( om
0.1 nM o 1 mM) o d ugs and compounds wi h an ipsycho ic, an icholine gic, na co ic,
and analgesic p ope ies (olanzapine, clozapine, desclozapine, luphenazine, pen azocine,
PB 28, DXT, DMT, donepezil, BD 1047, PPCC, and NE 100) in he p esence and absence
o 50
µ
M dUQ. The eac ion s a ed by he addi ion o memb ane homogena es, and NBT
oxida ion was measu ed e e y 5 min spec opho ome ically a 595 nM in a Mul iskan
FC mic o i e pla e eade (The mo Scien i ic
®
, Wal ham, MA, USA). Biochemical da a on
NADH oxido educ ase-e oked supe oxide p oduc ion we e p esen ed as a pe cen age o
basal ac i i y in he absence o he d ug unde s udy, wi h o wi hou decylubiquinone.
E e y CMMA had h ee eplica es o each issue, and all he expe imen s we e pe o med
in duplica e.
2.5. De e mina ion o Tissue-Speci ic E ec s o D ugs on Supe oxide Fo ma ion T igge ed by
NADH Using Human CMMAs
The NADH-ubiquinone oxido educ ase ac i i y was pe o med using cell memb ane
mic oa ays om a human issue collec ion (hea , li e , jejunum, duodenum, enal medulla,
enal co ex, ad enal gland, adipose issue, spleen, and lung). CMMAs we e incuba ed in
he p esence o 0.35 mM NADH and 0.05 mg/mL NBT in phospha e bu e (5 mM; pH 7.4)
o 1 h a 25
◦
C, wi h he compounds unde s udy (olanzapine, luphenazine, pen azocine,
PB28, and ne azodone) a 30
µ
M in he p esence o absence o 50
µ
M dUQ. The eac ion
was s a ed by he addi ion o he eagen s o he CMMAs. A e he incuba ion ime, he
eac ion was s opped by a dipping in dH
2
O. Once d ied, he CMMA colo signal was
acqui ed wi h an Epson V750 p o scanne , and digi al images we e analyzed wi h he
Biomedicines 2022,10, 980 5 o 16
so wa e Adobe Pho oshop CS5 (Adobe Sys ems Inco po a ed, Moun ain View, CA, USA)
and quan i ied using so wa e ImageScanne (IMG Pha ma S.L, De io, Spain).
2.6. De e mina ion o Tissue-Speci ic E ec on Supe oxide Fo ma ion Induced by Succina e Using
Human CMMAs
Succina e dehyd ogenase ac i i y was pe o med on cell memb ane mic oa ays om
a human issue collec ion (hea , li e , jejunum, duodenum, enal medulla, enal co ex,
ad enal gland, adipose issue, spleen, and lung). CMMAs we e incuba ed in he p esence
o 1 mM succina e, 0.05 mg/mL NBT, and 50
µ
M dUQ in phospha e bu e (5 mM; pH 7.4)
o 16 h a 25
◦
C. The eac ion was s a ed by he addi ion o he eagen s o he CMMAs.
A e he incuba ion ime, he eac ion was s opped by a dipping in dH
2
O. Once d ied,
he CMMA colo signal was acqui ed wi h an Epson V750 p o scanne , and digi al images
we e analyzed wi h he so wa e Adobe Pho oshop CS5 (Adobe Sys ems Inco po a ed,
Moun ain View, CA, USA) and quan i ied using so wa e ImageScanne (IMG Pha ma S.L,
De io, Spain).
2.7. De e mina ion o D ug Tissue-Speci ic E ec on Cy och ome C Oxidase Ac i i y
Cy och ome c oxidase ac i i y was assayed on cell memb ane mic oa ays om a
human issue collec ion. CMMAs we e incuba ed in he p esence o 1.3 mM o DAB and
0.01% o cy och ome c in phospha e bu e (0.1 M; pH 7.4) o 16 h a 37
◦
C in da kness.
A e he incuba ion ime, he eac ion was s opped by a dipping in dH
2
O. Once d ied,
he CMMA colo signal was acqui ed wi h an Epson V750 p o scanne , and digi al images
we e analyzed wi h he so wa e Adobe Pho oshop CS5 (Adobe Sys ems Inco po a ed,
Moun ain View, CA, USA) and quan i ied using so wa e ImageScanne (IMG Pha ma S.L,
De io, Spain).
2.8. Da a Analysis and No maliza ion
Da a handling and analysis we e ca ied ou using Excel and G aphPad so wa e
( e sion 9.2). The iden i ica ion o ou lie s was ca ied ou applying he ollowing o mulas:
CV =SD
X
Y1=X−DF ×SD
Y2=X+DF ×SD
SD = s anda d de ia ion; DF = de ia ion ac o ; and CV = a ia ion coe icien .
Poin s we e iden i ied as ou lie s and excluded i he a ia ion coe icien (CV) was
highe han 0.15, Y
1
was highe han he poin analyzed, o Y
2
was lowe han he poin
examined. We used a de ia ion ac o o 1 in ou analysis. In expe imen s pe o med wi h
bo ine hea memb anes homogena es, a nonlinea analysis (log (agonis ) s. esponse and
log (inhibi o ) s. esponse) was pe o med.
Fo mic oa ays, he analysis da a ob ained we e no malized o he amoun o o al
p o ein and we e exp essed as means o independen da a poin s
±
S.E.M. The no mali y o
he da a was es ed using Shapi o–Wilk s a is ical es wi h
α
: 0.05. Fo Gaussian dis ibu ed
da a, a s a is ical analysis was pe o med by one-way, wo- ailed ANOVA wi h Tukey’s
mul iple compa ison es . To analyze nonpa ame ical da a, he K uskal–Wallis es wi h
Dunn’s mul iple compa ison es was pe o med. S a is ical di e ences we e indica ed by
p- alues ≤0.05.
3. Resul s
3.1. P o ocol Op imiza ion o Supe oxide Fo ma ion in Bo ine Hea Memb anes Homogena es
The e ec o he di e en compounds s udied ela ing o supe oxide o ma ion was
de e mined in isola ed bo ine hea memb anes ollowing he p o ocol desc ibed in he
ma e ials and me hods sec ion.
Biomedicines 2022,10, 980 6 o 16
Fi s ly, succina e dehyd ogenase ac i i y assays we e pe o med de e mining DCIP
educ ion (Figu e S1), and NADH consump ion assays (Figu e S2) we e pe o med o
ensu e ha memb anes we e p ese ed and unc ional. A e alida ion, he o ma ion
o supe oxide in hese memb anes was e alua ed by de e mining he educ ion o NBT
in he p esence o NADH as subs a e, wi h o wi hou decylubiquinone (Figu e S3) o
s udy NADH dehyd ogenase ac i i y (complex I). As expec ed, he p esence o he de-
cylubiquinone anspo e inc eased he supe oxide o ma ion a e. Mo eo e , mETC
supe oxide gene a ion was es ed in he p esence o o enone (5
µ
M) and an imycin A
(5
µ
M) as inhibi o s o complexes I and III, espec i ely (Figu e S3). A e wa ds, NADH
dehyd ogenase ac i i y assays we e pe o med wi h di e en concen a ions o hese
inhibi o s. To de e mine he maximum e ec , dose– esponse cu es we e plo ed, and
nonlinea eg ession was used o each one (Figu e 2A, Table 1). Mo eo e , supe oxide o -
ma ion eloci ies we e de e mined o each concen a ion; a dose– esponse cu e was hen
plo ed, and nonlinea eg ession was used in o de o calcula e he maximum supe oxide
o ma ion eloci y (Figu e 2B, Table 1).
Biomedicines 2022, 10, x FOR PEER REVIEW 6 o 18
3. Resul s
3.1. P o ocol Op imiza ion o Supe oxide Fo ma ion in Bo ine Hea Memb anes Homogena es
The e ec o he di e en compounds s udied ela ing o supe oxide o ma ion was
de e mined in isola ed bo ine hea memb anes ollowing he p o ocol desc ibed in he
ma e ials and me hods sec ion.
Fi s ly, succina e dehyd ogenase ac i i y assays we e pe o med de e mining DCIP
educ ion (Figu e S1), and NADH consump ion assays (Figu e S2) we e pe o med o en-
su e ha memb anes we e p ese ed and unc ional. A e alida ion, he o ma ion o
supe oxide in hese memb anes was e alua ed by de e mining he educ ion o NBT in
he p esence o NADH as subs a e, wi h o wi hou decylubiquinone (Figu e S3) o s udy
NADH dehyd ogenase ac i i y (complex I). As expec ed, he p esence o he decylubiq-
uinone anspo e inc eased he supe oxide o ma ion a e. Mo eo e , mETC supe oxide
gene a ion was es ed in he p esence o o enone (5 μM) and an imycin A (5 μM) as in-
hibi o s o complexes I and III, espec i ely (Figu e S3). A e wa ds, NADH dehyd ogen-
ase ac i i y assays we e pe o med wi h di e en concen a ions o hese inhibi o s. To
de e mine he maximum e ec , dose– esponse cu es we e plo ed, and nonlinea eg es-
sion was used o each one (Figu e 2A, Table 1). Mo eo e , supe oxide o ma ion eloci-
ies we e de e mined o each concen a ion; a dose– esponse cu e was hen plo ed, and
nonlinea eg ession was used in o de o calcula e he maximum supe oxide o ma ion
eloci y (Figu e 2B, Table 1).
–7 –5 –3
100
120
140
160
Log [Inhibi o ] (M)
Supe oxide o ma ion media ed by
NADH dehyd ogenase
Ro enone
An imycin A
–9 –7 –5 –3
60
80
100
120
140
Log [Inhibi o ] (M)
Supe oxide o ma ion speed media ed
by NADH dehyd ogenase
Ro enone
An imycin A
A) B)
Figu e 2. (A) Concen a ion– esponse cu es ob ained om he maximum supe oxide o ma ion
e oked by NADH in he p esence o mi ochond ial elec on anspo chain inhibi o s o he com-
plex I ( o enone) and III (An imycin A). (B) Supe oxide o ma ion a e e oked by NADH in he
p esence o o enone and an imycin A de e mined om he lineal ange o he kine ic assays. (A)
Nonlinea eg ession was pe o med wi h log(agonis ) s. esponse model and leas squa es eg es-
sion as i ing me hod. (B) Nonlinea eg ession was pe o med wi h log(inhibi o ) s. esponse
model o o enone and log(agonis ) s. esponse model o an imycin A. In bo h cases, leas squa es
eg ession was used as i ing me hod.
Table 1. Loga i hms o hal -maximum e ec i e o inhibi o y concen a ions (pEC50 o pIC50), max-
imum supe oxide o ma ion (Emax), and maximum supe oxide o ma ion eloci y (Vmax) de e -
mined om concen a ion– esponse cu es o each speci ic inhibi o .
Supe oxide Fo ma ion
Maximum Amoun
P oduc ion Ra e
Ro enone
pEC50
−7.7 ± 0.23
42.79 ± 4.88
pIC50
−7.47 ± 0.15
41.90 ± 2.94
Emax
Vmax
An imycin A
pEC50
−5.94 ± 0.14
34.20 ± 2.29
pEC50
−6.30 ± 0.35
21.04 ± 3.35
Emax
Vmax
Figu e 2.
(
A
) Concen a ion– esponse cu es ob ained om he maximum supe oxide o ma ion
e oked by NADH in he p esence o mi ochond ial elec on anspo chain inhibi o s o he complex
I ( o enone) and III (An imycin A). (
B
) Supe oxide o ma ion a e e oked by NADH in he p esence
o o enone and an imycin A de e mined om he lineal ange o he kine ic assays. (
A
) Nonlinea
eg ession was pe o med wi h log(agonis ) s. esponse model and leas squa es eg ession as i ing
me hod. (
B
) Nonlinea eg ession was pe o med wi h log(inhibi o ) s. esponse model o o enone
and log(agonis ) s. esponse model o an imycin A. In bo h cases, leas squa es eg ession was used
as i ing me hod.
Table 1.
Loga i hms o hal -maximum e ec i e o inhibi o y concen a ions (pEC50 o pIC50), maxi-
mum supe oxide o ma ion (Emax), and maximum supe oxide o ma ion eloci y (Vmax) de e mined
om concen a ion– esponse cu es o each speci ic inhibi o .
Supe oxide Fo ma ion
Maximum Amoun P oduc ion Ra e
Ro enone pEC50 −7.7 ±0.23 pIC50 −7.47 ±0.15
Emax 42.79 ±4.88 Vmax 41.90 ±2.94
An imycin A pEC50 −5.94 ±0.14 pEC50 −6.30 ±0.35
Emax 34.20 ±2.29 Vmax 21.04 ±3.35
Bo h inhibi o s inc eased he o al amoun o supe oxide p oduced (42.8% o enone
s. 34.2% an imycin A) bu wi h di e en po ency (Figu e 2A, Table 1). By con as , hei
e ec s on eac ion eloci y we e subs an ially di e en as o enone educed i , whe eas
an imycin A inc eased i (Figu e 2B, Table 1).
Maximum eloci ies we e calcula ed by a nonlinea model using a eloci y s. log
[inhibi o ] cu e (Figu e 2B). The a es a each concen a ion we e achie ed om a lineal
Biomedicines 2022,10, 980 7 o 16
ange o supe oxide o ma ion assays (Figu e S3) de e mining he slopes o e e y di e en
inhibi o concen a ion. To calcula e pha macological pa ame e s, we used hese eloci ies
and he maximum e ec (Emax) ob ained om he kine ic dose– esponse cu es (Figu e 2B).
3.2. E ec o D ugs and Compounds on Supe oxide Fo ma ion in Isola ed Bo ine Hea Memb anes
The capaci y o supe oxide o ma ion o 12 di e en medicines wi h an ipsycho ic,
an icholine gic, na co ic, and analgesic p ope ies was assessed: BD1047, PB28, NE100, and
PPCC as sigma ecep o an agonis s; luphenazine, pen azocine, olanzapine, clozapine, and
desclozapine as i s and second-gene a ion an ipsycho ics; and N, N-dime hyl yp amine
(DMT), donepezil, and dex ome ho phan (DXT) as o he d ugs wi h neu ological ac ions.
An inc ease in eac i e oxygen species o ma ion was obse ed wi h ce ain d ugs,
namely, NE100 (sigma 1 ecep o an agonis ), pen azocine (analgesic), and PB28 (sigma 2
agonis ) wi h he highe maximum e ec o 60.1%, 45.5%, and 43.4%, espec i ely (Figu e 3;
Table 2). On he o he hand, PPCC (sigma 1 ecep o agonis ) and luphenazine (an ipsy-
cho ic) eached an Emax o 35.4% and 36.8%, espec i ely. Finally, he ones wi h a lowe
Emax we e BD1047 (sigma 1 ecep o an agonis ), olanzapine (an ipsycho ic), and DXT
(an i ussi e). DMT (sigma 1 ecep o agonis ), clozapine (an ipsycho ic), and desclozapine
(an ipsycho ic) did no p omo e any e ec .
Biomedicines 2022, 10, x FOR PEER REVIEW 7 o 18
Bo h inhibi o s inc eased he o al amoun o supe oxide p oduced (42.8% o enone
s. 34.2% an imycin A) bu wi h di e en po ency (Figu e 2A, Table 1). By con as , hei
e ec s on eac ion eloci y we e subs an ially di e en as o enone educed i , whe eas
an imycin A inc eased i (Figu e 2B, Table 1).
Maximum eloci ies we e calcula ed by a nonlinea model using a eloci y s. log
[inhibi o ] cu e (Figu e 2B). The a es a each concen a ion we e achie ed om a lineal
ange o supe oxide o ma ion assays (Figu e S3) de e mining he slopes o e e y di e -
en inhibi o concen a ion. To calcula e pha macological pa ame e s, we used hese e-
loci ies and he maximum e ec (Emax) ob ained om he kine ic dose– esponse cu es
(Figu e 2B).
3.2. E ec o D ugs and Compounds on Supe oxide Fo ma ion in Isola ed Bo ine Hea
Memb anes
The capaci y o supe oxide o ma ion o 12 di e en medicines wi h an ipsycho ic,
an icholine gic, na co ic, and analgesic p ope ies was assessed: BD1047, PB28, NE100,
and PPCC as sigma ecep o an agonis s; luphenazine, pen azocine, olanzapine, clozap-
ine, and desclozapine as i s and second-gene a ion an ipsycho ics; and N, N-dime hyl-
yp amine (DMT), donepezil, and dex ome ho phan (DXT) as o he d ugs wi h neu o-
logical ac ions.
An inc ease in eac i e oxygen species o ma ion was obse ed wi h ce ain d ugs,
namely, NE100 (sigma 1 ecep o an agonis ), pen azocine (analgesic), and PB28 (sigma 2
agonis ) wi h he highe maximum e ec o 60.1%, 45.5%, and 43.4%, espec i ely (Figu e
3; Table 2). On he o he hand, PPCC (sigma 1 ecep o agonis ) and luphenazine (an i-
psycho ic) eached an Emax o 35.4% and 36.8%, espec i ely. Finally, he ones wi h a
lowe Emax we e BD1047 (sigma 1 ecep o an agonis ), olanzapine (an ipsycho ic), and
DXT (an i ussi e). DMT (sigma 1 ecep o agonis ), clozapine (an ipsycho ic), and
desclozapine (an ipsycho ic) did no p omo e any e ec .
–7 –6 –5 –4 –3
100
120
140
160
Log [D ug] (M)
Supe oxide o ma ion media ed by
NADH dehyd ogenase
PENTAZOCINE
BD1047
DXT
–7 –6 –5 –4 –3
100
120
140
160
Log [D ug] (M)
Supe oxide o ma ion media ed by
NADH dehyd ogenase
OLANZAPINE
DESCLOZAPINE
–7 –6 –5 –4 –3
100
120
140
160
Log [D ug] (M)
Supe oxide o ma ion media ed by
NADH dehyd ogenase
PB28
NE100
PPCC
DMT
–7 –6 –5 –4 –3
100
120
140
160
Log [D ug] (M)
Supe oxide o ma ion media ed by
NADH dehyd ogenase
CLOZAPINE
FLUPHENAZINE
DONEPEZIL
Figu e 3. Concen a ion– esponse cu es o supe oxide o ma ion in isola ed bo ine hea mem-
b anes induced by NADH dehyd ogenase modula ion wi h pen azocine, BD1047, DXT, PB28,
NE100, PPCC, DMT, olanzapine, desclozapine, clozapine, luphenazine, and donepezil. The
Figu e 3.
Concen a ion– esponse cu es o supe oxide o ma ion in isola ed bo ine hea memb anes
induced by NADH dehyd ogenase modula ion wi h pen azocine, BD1047, DXT, PB28, NE100, PPCC,
DMT, olanzapine, desclozapine, clozapine, luphenazine, and donepezil. The supe oxide o ma ion
p omo ed by NADH dehyd ogenase is ep esen ed in pe cen ages e sus he ac i i y measu ed in
absence o he es ed d ug. Nonlinea eg ession was pe o med wi h he log (agonis ) s. esponse
( h ee pa ame e s) model and leas squa es eg ession as i ing me hod.
Rega ding he a e o eac i e oxygen species o ma ion, mos o he d ugs s udied
educed he a e a high concen a ions, excep o DXT, olanzapine, clozapine, and de-
sclozapine, which did no a ec i (Figu e S4). The kine ic pa ame e s we e de e mined,
and a educ ion in he a e o supe oxide o ma ion p omo ed by luphenazine and PB28
was obse ed, eaching 51.9% and 34.0%, espec i ely. Pen azocine, PPCC, and donepezil
also p omo ed a a e educ ion o 23.9%, 21.6%, and 17.3%, espec i ely (Table S1). By
Biomedicines 2022,10, 980 8 o 16
con as , clozapine, desclozapine, olanzapine, DMT, DXT, and BD1047 did no signi ican ly
change he a e o supe oxide o ma ion e oked by NADH dehyd ogenase.
Table 2.
Po ency (pEC50) and maximum e ec (Emax) calcula ed o each d ug on supe oxide
o ma ion e oked by NADH dehyd ogenase ac i i y in isola ed bo ine hea memb anes.
pEC50 Emax (%) pEC50 Emax (%)
PB 28 −5.4 ±0.2 43.4 ±3.0 Pen zazocine −3.9 ±0.3 45.5 ±11.1
NE 100 −3.6 ±0.3 60.1 ±23.1 Donepezil UD UD
PPCC −4.5 ±0.3 35.4 ±5.3
Fluphenazine
−4.6 ±0.3 36.8 ±4.9
BD 1047 −6.3 ±0.4 20.6 ±4.1 Clozapine UD UD
DMT UD UD Olanzapine −5.5 ±0.4 15.7 ±3.1
DXT −3.9 ±0.3 19.0 ±5.2 Desclozapine −3.2 ±1.0 UD
UD = unde e mined.
3.3. Tissue-Speci ic E ec s o D ugs on Supe oxide Fo ma ion Using CMMAs o Human Tissues
To ensu e ha he immobilized memb anes ha cons i u e he CMMAs we e unc ional
and he mi ochond ial memb anes we e p ese ed, he supe oxide o ma ion igge ed by
complex I and complex II subs a es and he cy och ome c Oxidase ac i i y we e assayed
(Figu e 4).
Biomedicines 2022, 10, x FOR PEER REVIEW 8 o 18
supe oxide o ma ion p omo ed by NADH dehyd ogenase is ep esen ed in pe cen ages e sus he
ac i i y measu ed in absence o he es ed d ug. Nonlinea eg ession was pe o med wi h he log
(agonis ) s. esponse ( h ee pa ame e s) model and leas squa es eg ession as i ing me hod.
Table 2. Po ency (pEC50) and maximum e ec (Emax) calcula ed o each d ug on supe oxide o -
ma ion e oked by NADH dehyd ogenase ac i i y in isola ed bo ine hea memb anes.
pEC50
Emax (%)
pEC50
Emax (%)
PB 28
−5.4 ± 0.2
43.4 ± 3.0
Pen zazocine
−3.9 ±0.3
45.5 ± 11.1
NE 100
−3.6 ± 0.3
60.1 ± 23.1
Donepezil
UD
UD
PPCC
−4.5 ± 0.3
35.4 ± 5.3
Fluphenazine
−4.6 ± 0.3
36.8 ± 4.9
BD 1047
−6.3 ± 0.4
20.6 ± 4.1
Clozapine
UD
UD
DMT
UD
UD
Olanzapine
−5.5 ± 0.4
15.7 ± 3.1
DXT
−3.9 ± 0.3
19.0 ± 5.2
Desclozapine
−3.2 ± 1.0
UD
* UD = unde e mined.
Rega ding he a e o eac i e oxygen species o ma ion, mos o he d ugs s udied
educed he a e a high concen a ions, excep o DXT, olanzapine, clozapine, and
desclozapine, which did no a ec i (Figu e S4). The kine ic pa ame e s we e de e mined,
and a educ ion in he a e o supe oxide o ma ion p omo ed by luphenazine and PB28
was obse ed, eaching 51.9% and 34.0%, espec i ely. Pen azocine, PPCC, and donepezil
also p omo ed a a e educ ion o 23.9%, 21.6%, and 17.3%, espec i ely (Table S1). By
con as , clozapine, desclozapine, olanzapine, DMT, DXT, and BD1047 did no signi i-
can ly change he a e o supe oxide o ma ion e oked by NADH dehyd ogenase.
3.3. Tissue-Speci ic E ec s o D ugs on Supe oxide Fo ma ion Using CMMAs o Human Tissues
To ensu e ha he immobilized memb anes ha cons i u e he CMMAs we e unc-
ional and he mi ochond ial memb anes we e p ese ed, he supe oxide o ma ion ig-
ge ed by complex I and complex II subs a es and he cy och ome c Oxidase ac i i y we e
assayed (Figu e 4).
Figu e 4. Rep esen a i e image o human CMMAs showing he immobilized p o ein (B ad o d), he
ac i i y o he NADH dehyd ogenase (NADH), and succina e dehyd ogenase (SDH) oge he wi h
he ac i i y o ci och ome c oxidase (CcO) o he mi ochond ial elec on anspo chain.
The selec ion o d ugs es ed on human CMMAs was based on he esul s o he ex-
pe imen s wi h bo ine hea memb anes, he in o ma ion a ailable in he li e a u e, a link
be ween he ad e se e ec s o hese d ugs, and mi ochond ial oxida i e s ess [62,63]. The
d ugs selec ed we e luphenazine, pen azocine, olanzapine, PB28, and ne azodone. In he
human hea , all d ugs excep ne azodone p omo ed an inc ease in supe oxide o ma ion
Figu e 4.
Rep esen a i e image o human CMMAs showing he immobilized p o ein (B ad o d), he
ac i i y o he NADH dehyd ogenase (NADH), and succina e dehyd ogenase (SDH) oge he wi h
he ac i i y o ci och ome c oxidase (CcO) o he mi ochond ial elec on anspo chain.
The selec ion o d ugs es ed on human CMMAs was based on he esul s o he
expe imen s wi h bo ine hea memb anes, he in o ma ion a ailable in he li e a u e, a
link be ween he ad e se e ec s o hese d ugs, and mi ochond ial oxida i e s ess [
62
,
63
].
The d ugs selec ed we e luphenazine, pen azocine, olanzapine, PB28, and ne azodone. In
he human hea , all d ugs excep ne azodone p omo ed an inc ease in supe oxide o ma-
ion in he absence o dUQ (13.8% olanzapine <49.1% luphenazine <72.5%
PB28 < 85.0%
pen azocine). In he p esence o he dUQ elec on anspo e , olanzapine inc eased he
supe oxide o ma ion om 13.8% o 52.0%, while luphenazine, PB28, and pen azocine
educed i ( om 49.1% o 8.3% o luphenazine; 72.5% o
−
4.1% o PB28; and 85.0% o
46.5% o pen azocine) (Figu es 5and 6).
Rega ding o he issues, olanzapine signi ican ly enhanced he supe oxide p oduc ion
in li e , duodenum, ad enal gland, and enal medulla. The p esence o dUQ did no
al e he e ec p omo ed by olanzapine alone in any o he issues, excep in spleen and
lung, whe e a signi ican inc ease in supe oxide o ma ion was obse ed (Figu e 5A).
Fluphenazine only induced a signi ican inc ease in supe oxide p oduc ion in li e and
Biomedicines 2022,10, 980 9 o 16
hea , while in all o he issues, i did no enhance and e en seemed o dec ease i . These
ac ions we e blocked by dUQ o e en e e ed in some issues, such as spleen (Figu e 5B).
Pen azocine e oked a subs an ial inc ease in supe oxide p oduc ion in hea , li e , and
jejunum issues. A educ ion in o he issues, such as enal medulla, ad enal gland, o
spleen, was achie ed wi h his compound in he absence o dUQ, while he p esence o he
anspo e e e ed hese ac ions (Figu e 6A). PB28 inc eased he supe oxide o ma ion in
hea , li e , jejunum, enal medulla, adipose issue, and spleen. Howe e , he coincuba ion
o PB28 wi h dUQ signi ican ly blocked i in hea , jejunum, enal medulla, and adipose
issue (Figu e 6B). Finally, ne azodone caused an inc ease in supe oxide o ma ion in li e ,
jejunum, duodenum, and lung issues in he absence o dUQ, while in he p esence o his
elec on anspo e , i was no a ec ed, excep in he case o he duodenum, whe e i was
also enhanced (Figu e 6C). In enal medulla, enal co ex, ad enal gland, and adipose issue,
he coincuba ion o ne azodone wi h dUQ e oked an inc ease in supe oxide o ma ion. In
con as , in hea issue, a educ ion o supe oxide o ma ion was obse ed (Figu e 6C).
Biomedicines 2022, 10, x FOR PEER REVIEW 9 o 18
in he absence o dUQ (13.8% olanzapine <49.1% luphenazine <72.5% PB28 < 85.0% pen-
azocine). In he p esence o he dUQ elec on anspo e , olanzapine inc eased he su-
pe oxide o ma ion om 13.8% o 52.0%, while luphenazine, PB28, and pen azocine e-
duced i ( om 49.1% o 8.3% o luphenazine; 72.5% o −4.1% o PB28; and 85.0% o
46.5% o pen azocine) (Figu es 5 and 6).
Hea
Li e
Jejunum
Duodenum
Renal medulla
Renal co ex
Ad enal gland
Adipode issue
Spleen
Lung
-100
-50
0
50
100
150
400
Supe oxide o ma ion media ed by
NADH dehyd ogenase (% s con ol)
Olanzapine
*
*
####
#
*
A)
D ug + dUQ
D ug
Hea
Li e
Jejunum
Duodenum
Renal medulla
Renal co ex
Ad enal gland
Adipode issue
Spleen
Lung
-100
-50
0
50
100
150
400
Supe oxide o ma ion media ed by
NADH dehyd ogenase (% s con ol)
Fluphenazine
***
#
##
**
#
**
###
B)
Figu e 5. (A) NADH-media ed supe oxide o ma ion p omo ed by olanzapine in di e en human
issues in he p esence and absence o decylubiquinone. (B) NADH-media ed supe oxide o ma ion
p omo ed by luphenazine in di e en human issues in he p esence and absence o decylubiqui-
none. Resul s exp essed as pe cen ages o inc ease e sus he basal ac i i y wi hou he d ug (mean
± SEM). Shapi o–Wilk es was pe o med o es no mali y. Fo he issues ha p esen a Gaussian
dis ibu ion, one-way ANOVA s a is ical es wi h α: 0.05 was pe o med. Fo issues wi hou a no -
mal dis ibu ion, K uskal–Wallis s a is ical es wi h α: 0.05 was pe o med: * p < 0.05; ** p < 0.01; ***
p < 0.001 d ug s. con ol; # p < 0.05; ## p < 0.01; ### p < 0.001; and #### p < 0.0001 d ug alone s. d ug
+ dUQ.
Figu e 5.
(
A
) NADH-media ed supe oxide o ma ion p omo ed by olanzapine in di e en human
issues in he p esence and absence o decylubiquinone. (
B
) NADH-media ed supe oxide o ma ion
p omo ed by luphenazine in di e en human issues in he p esence and absence o decylubiquinone.
Resul s exp essed as pe cen ages o inc ease e sus he basal ac i i y wi hou he d ug (
mean ±SEM
).
Shapi o–Wilk es was pe o med o es no mali y. Fo he issues ha p esen a Gaussian dis ibu ion,
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α
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