Co esponding au ho : Lamidi Waheed B. Olaniyan
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion Liscense 4.0.
Mix u e e ec s o h ee analgesics on biochemical indices in a enal mi ochond ial
ac ion
Lamidi Waheed B. Olaniyan 1, *, Olajumoke Qud a Owolabi 1 and Tao ik R. Gbadamosi 2
1 Depa men o Biochemis y, Facul y o Pu e and Applied Sciences Ladoke Akin ola Uni e si y o Technology, Ogbomoso
Nige ia.
2 Depa men o Biochemical Science, Fede al Poly echnic Ede, Nige ia.
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 258-267
Publica ion his o y: Recei ed on 16 Augus 2025; e ised on 25 Sep embe 2025; accep ed on 27 Sep embe 2025
A icle DOI: h ps://doi.o g/10.30574/gscbps.2025.32.3.0370
Abs ac
Humans a e exposed o di e en mix u es o biologically ac i e chemicals, unin en ionally o in en ionally o example
by medica ions o h ough oods. The exposu e isks o hese chemical combina ions a e a om being unde s ood.
Acco ding o he Wo ld Heal h O ganiza ion, e idence has shown ha chemicals wi hou ac ion a indi idual le els may
ac addi i ely and cause p oblems. Thus, human exposu e o a mix u e could p oduce biological esponses which may
no be p edic able when used as single compounds. D ug combina ion is a common in e en ion in pain managemen ,
especially in pa ien s wi h como bidi ies and complex pain synd omes. Aspi in, ca eine and pa ace amol combina ion
has been used in pain managemen bu hei oxicological implica ions ha e no been clea ly wo ked ou . The
widesp ead use o analgesics such as pa ace amol, aspi in and indome hacin o pain managemen has aised conce ns
abou hei po en ial ad e se e ec s, pa icula ly on i al o gans such as he kidneys. This s udy aimed o in es iga e
he mix u e e ec o h ee commonly p esc ibed analgesics on he kidney mi ochond ial ac ion o Sp ague Dawley
a s. They we e o ally exposed in 2 mL wa e o aspi in, pa ace amol and indome hacin singly and in combina ion a
hei espec i e he apeu ic doses daily o 14 days. The con ol a s ecei ed wa e and ood only. Ce ain biochemical
pa ame e s we e assessed in he a s’ kidney mi ochond ial ac ion by spec opho ome ic echniques. The s a is ical
analysis o da a was done using G aphpad p ism so wa e. Da a we e exp essed as Mean ± SEM. The means we e
subjec ed o one-way analysis o a iance using Tukey as a pos -hoc analysis. Values we e conside ed s a is ically
signi ican a p<0.05. The ac i i y o he supe oxide dismu ase unde he Mix (d ug combina ion g oup) inc eased
signi ican ly when compa ed wi h he espec i e enzyme ac i i y unde he indi idual d ugs. Ex en o lipid
pe oxida ion, concen a ions o non-p o ein sulphyd yl (NPSH) and c ea inine we e lowe in he Mix g oup ela i e o
he es g oups. Aspi in g oup eco ded he leas lipid pe oxida ion and NPSH concen a ion while indome hacin g oup
he highes . The highes c ea inine concen a ion was eco ded among a s dosed pa ace amol. The p o-oxidan abili y
o he d ugs and hence enal oxici y appea o be in he o de Pa ace amol ˃ Indome hacine ˃ Combina ion dose ˃
Aspi in. Aspi in e ec and p esumably mechanism o i s oxici y may be an agonis ic o he ac i i y o he es o he
d ugs. These indings indica ed ha he d ug mix u e was less p o-oxidan han he indi idual d ugs a hei he apeu ic
doses in he a kidney.
Keywo ds: Analgesics; Combina ion E ec s; Oxida i e S ess; Mi ochond ial Dys unc ion; Renal Toxici y
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259
G aphical abs ac
1. In oduc ion
Human and animals a e exposed o di e se biologically ac i e chemicals om a ious en i onmen al media sou ced
om ag icul u e, indus y, medical acili ies, and household was es Al hough mos an h opogenic compounds in he
en i onmen a e always p esen a e y low concen a ions, “in en ional” exposu e o hese compounds such as by
medica ion [1] occu s a highe concen a ions and can as well lead o bioaccumula ion in he a y issues engende ing
oxici y [2, 3]. Unin en ional human exposu e o pollu an s in he en i onmen al ma ices (ai , soil and wa e ) in ol es
complex chemical mix u es o compounds o di e se na u e. The oxicological e ec s o compounds mix u e can esul
in ei he independence ( esponse addi i i y) o dose addi ion. Concen a ion (o dose) addi ion has been used in
associa ion wi h mix u es o simila ac ing chemicals such as hose in e ac ing wi h ER, while independen ac ion has
been employed o di e en ac ing compounds, including compounds in a mix u e ha in e ac wi h dissimila
ecep o s o o he molecula a ge s. Thus, human exposu e o a mix u e could p oduce biological esponses which may
no be p edic able when using single compounds [4]. Acco ding o he Wo ld Heal h O ganiza ion [5], he e is eme ging
e idence ha many chemicals may ac addi i ely and, each a le els wi hou indi idual e ec , could ac oge he o cause
heal h p oblems.
Pain is a common symp om ha can signi ican ly impai an indi idual's quali y o li e and p oduc i i y. As a esul , he
use o analgesics, including non-s e oidal an i-in lamma o y d ugs (NSAIDs) and he opioids, has become a s anda d
p ac ice o pain managemen . D ug combina ion is a common in e en ion in pain managemen , especially in pa ien s
wi h como bidi ies and complex pain synd omes [6]. Dose combina ion o ace ylsalicyclic acid (aspi in), pa ace amol
and ca eine is commonly used mix analgesic [7]. Howe e , he widesp ead use o analgesics has aised conce ns abou
hei po en ial ad e se e ec s, pa icula ly on i al o gans such as he kidneys [8]. Any d ug-induced damage o he
kidneys can lead o signi ican heal h complica ions, including acu e and ch onic enal ailu e. Mi ochond ia a e he
cellula o ganelles esponsible o ene gy p oduc ion h ough oxida i e phospho yla ion. Mi ochond ial dys unc ion is
a key con ibu o o se e al pa hological condi ions, including neu odegene a i e diso de s, cance , and ca dio ascula
diseases. The kidney is a highly ene gy-demanding o gan ha elies hea ily on mi ochond ial unc ion o main ain i s
physiological unc ions [9]. Consequen ly, d ug-induced mi ochond ial dys unc ion in he kidney can lead o enal inju y
and dys unc ion. Howe e , he po en ial ad e se e ec s o d ug combina ions on mi ochond ial unc ion in he kidney
emain la gely unexplo ed. The e o e, unde s anding he e ec s o analgesic d ug combina ions on enal mi ochond ial
unc ion is c i ical o he de elopmen o sa e pain managemen s a egies. In his s udy, we aimed o deciphe he
mix u e e ec o h ee commonly p esc ibed analgesics on he kidney mi ochond ial ac ion o Sp ague a s. The
selec ed analgesics included indome hacin a non-s e oidal an i-in lamma o y d ug (NSAID) and mechanis ically a non-
selec i e cyclooxygenase (COX) inhibi o [10]. Indome hacin is a de i a i e o indoleace ic acid and iodoace ic acid.
Unlike indome hacin, pa ace amol o ace aminophen is no a membe o NSAID bu is highly selec i e o
cyclooxygenase-2 wi h a weak an i-in lamma o y ac i i y especially in he cen al ne ous sys em. I is equen ly
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 258-267
260
p esc ibed wi h o wi hou combina ion o pain managemen pa icula ly in pa ien s wi h ch onic pain synd omes
[1,11]. Pa ace amol e icacy and sa e y may be addi i e o syne gis ic when p esc ibed in combina ion wi h NSAIDs
[12]. Aspi in (Ace ylsalicylic acid) is a NSAID and widely used as an analgesic, an i-in lamma o y and an i-pla ele d ug
[13]. All he h ee medica ions a e known inhibi o s o p os aglandin syn hesis ia cyclooxygenase inhibi ion. Thei
an i-and ogenici y has also been epo ed in a oe al es is [14]. To assess he mix u e e ec o hese analgesics, we
e alua ed key pa ame e s associa ed wi h mi ochond ial unc ion and in eg i y and eac i e oxygen species (ROS)
gene a ion. We hypo hesized ha he simul aneous adminis a ion o he h ee analgesics a he apeu ic doses would
esul in a syne gis ic e ec leading o mi ochond ial dys unc ion and oxida i e s ess wi hin he kidney. The esul s o
his s udy may ha e signi ican implica ions o he sa e use o analgesics in pain managemen , pa icula ly in pa ien s
wi h enal como bidi ies.
2. Ma e ials and me hods
2.1. Reagen s
All eagen s used o his p ojec wo k we e o analy ical g ade ob ained ei he om Sigma Ald ich Company, Louis USA
o om ou acc edi ed con ac o s.
2.2. Expe imen al design
Appa en ly heal hy adul male Sp ague-Dawley a s (a e age weigh 150 ± 3.30 g) we e housed unde s anda d
condi ions o empe a u e (25ºC ± 2ºC) and humidi y (55% ± 10%) wi h a 12-hou ligh /da k cycle and ed pelle ed
die and wa e ad libi um. They we e acclima ized o 2 weeks be o e he expe imen s. The a s we e andomly di ided
in o i e g oups, each g oup con ained 5 a s. The i s g oup ( he con ol) consis ed o a s ed wi h basal die no
supplemen ed wi h he es d ugs. The second g oup was adminis e ed he apeu ic dose o aspi in equi alen o 16.7
mg/kg b w/day based on human adul weigh o 60 kg [15], he hi d g oup ecei ed indome hacin adminis e ed a
150 mg/kg b w/day [16] he apeu ic dose o gou y a h i is, while he ou h g oup go pa ace amol a a maximum
dose o 4 g/day o 0.067g/kg body weigh (b w)/day [17], he i h g oup was adminis e ed he mix doses o he d ugs
daily o 14 days. The d ug doses we e con ained in 2 mL dis illed wa e .
2.3. Sample collec ion
The animals we e sac i iced by decapi a ion on he 16 h day o adminis a ion. Blood samples we e allowed o clo and
he esul ing se a ca e ully aspi a ed wi hou aken up he cells. The kidneys we e ha es ed washed clean o connec i e
issues wi h ice-cold 0.15 M KCl and we e homogenized in ice-cold 0.25 M suc ose solu ion bu e ed wi h 40 mM T is.HCl
a pH 7.4 o in 0.02 M ice-cold EDTA in some expe imen s using Te lon-lined homogenize in all cases. Renal issue
mi ochond ial ac ion was ob ained by he me hod o Johnson and La dy [18] in a 0.25 M suc ose medium wi h 2 mg/ml
o albumin.
2.4. Biochemical Assays
Supe oxide dismu ase ac i i y was de e mined by using he me hod o Mis a and F ido ich [19] which is based on
inhibi ion o epineph ine au oxida ion by supe oxide anions a basic pH (10.2). F esh epineph ine solu ion was
p epa ed by dissol ing 13.7 mg o epineph ine in 250 mL o dis illed wa e . 1 mL o he sample was dilu ed o 10 mL
wi h dis illed wa e . The kine ics o he enzyme was ollowed by eading he abso bances a 480 nm a 30 s in e als
o a o al ime o 150 s agains blank made up o he eac ion mix u es less he sample. The change in abso bance was
inally calcula ed. Mola ex inc ion coe icien o he ad enoch ome a 480 nm was aken as 4020 M-1cm-1. The ac i i y
o he enzyme was de ined as he amoun o he enzyme ha ca ied ou he hal inhibi ion o he au oxida ion o
epineph ine o ad enoch ome unde he s a ed condi ions. Es ima ion o lipid pe oxida ion in he enal issue
homogena es was conduc ed by spec opho ome ic de e mina ion o malondialdehyde (MDA) concen a ion [20]. To
1 mL o he sample was added 0.5 ml 2.5% / HCl ollowed by 0.5 ml o hioba bi u ic acid solu ion (1% w/ in 50 mM
sodium hyd oxide). The eac ion mix u e was hea ed a 80°C o 30 min, cooled wi h unning wa e and he ch omogen
was ex ac ed wi h 2 mL o bu anol. The abso bance o he uppe laye was ead a 532 nm. The ex inc ion coe icien
was aken as 1.56 x 105 M-1cm-1 .
The sample olume was 0.5 mL. Concen a ion o non-p o ein bound sulphyd yl g oups (NPSH) was es ima ed as
explained by Sedlak and Lindsay [21]. The issues we e homogenized in ice cold 0.02 M disodium EDTA o his
in es iga ion. The abso bance o he esul ing solu ion was ead a 412 nm wi hin 5 mins o DTNB addi ion agains a
blank wi hou he homogena es. Mola ex inc ion coe icien o 2-ni o-5- hiobenzoic acid (TNB) is 14,150 M-1cm-1 [22].
P o ein concen a ion was de e mined by he spec opho ome ic me hod o B ad o d [23] in which Coomassie B illian
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Blue G-250 dye was dissol ed in 95% e hanol ollowed by addi ion o 85% phospho ic. The abso bance was ead a 595
nm. The p ocedu e was as con ained in Bioquan ki manual. A calib a ion cu e was cons uc ed om a se ies o
s anda d albumin concen a ions anging om 100 o 1000 µg/L. C ea inine concen a ion was de e mined by Ja e
eac ion as alkaline c ea inine pic a e [24; 25]. B ie ly, 1 mL. 0.04 M pic ic acid was mixed wi h 1 mL. 0.75 M sodium
hyd oxide o which 3 mL o he s anda d c ea inine (0.5 mg/mL p epa ed in 0.1 M HCl) was added. The o ange colou
was allowed o de elop in 20 minu es; he abso bance was ead agains dis illed wa e on a spec opho ome e a 520
nm. A se ies o c ea inine concen a ions was p epa ed and subjec ed o he es om which a calib a ion g aph was
cons uc ed. The unknown concen a ion o c ea inine om he p o ein- ee se um sample was ead o om he
s anda d g aph. Succina e dehyd ogenase ac i i y was de e mined acco ding o he con inuous spec opho ome ic
echnique o Munujos and co-wo ke s [26]. Change in abso bance o e 6 mins a 500 nm was calcula ed using he
ex inc ion coe icien o 19,300 M-1cm-1 [27].
2.5. S a is ical Analysis
The s a is ical analysis o da a was done using G aphpad p ism ( e sion 9). Da a we e exp essed as mean ± SEM and
we e subjec ed o one-way analysis o a iance (ANOVA) using Tukey- mul i- compa ison. Di e ence be ween means
was conside ed s a is ically signi ican a p < 0.05.
3. Resul s
The e ec o he d ug ea men s on he enal issues supe oxide dismu ase (SOD) speci ic ac i i y a e depic ed in Fig.
1. Gene ally he SOD ac i i y was signi ican ly (p ˂ 0.05) lowe in all ea ed animals han in he un ea ed con ol
animals. The e we e signi ican di e ences in SOD ac i i y be ween Mix (mix u e o he d ugs) and he es g oups
excep he pa ace amol g oup. The Aspi in g oup exhibi ed he lowes speci ic ac i i y o SOD. The malondialdehyde
(MDA) concen a ions we e signi ican ly inc eased among pa ace amol, indome hacin and Mix exposed a g oups
ela i e o he con ol (Fig. 2). The MDA concen a ion was highes in Indome hacin g oup whe eas aspi in induced he
leas quan i y. The di e ence in MDA concen a ion be ween aspi in and he con ol was signi ican . No s a is ically
signi ican di e ence was eco ded be ween pa ace amol exposed a s and he Mix a s; he e e se was he case
be ween Indome hacin and Mix a s.
Figu e 1 Supe oxide dismu ase ac i i y in a enal issues
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Figu e 2 Malondialdehyde concen a ion in he a s enal issues
The concen a ion o non-p o ein sulphyd yl (NPSH) was signi ican ly educed among all he ea ed a s when
compa ed wi h he Con ol (Fig. 3), whe e Mix showed he leas concen a ion among he exposed a s. All he exposed
g oups (aspi in, pa ace amol, indome hacin) showed signi ican inc ease in NPSH concen a ion when compa ed o he
Mix (Fig. 3).
Figu e 3 NPSH concen a ion in he enal issue o he a s
All he d ugs induced signi ican inc ease in c ea inine concen a ion compa ed wi h he Con ol (Fig. 4). Pa ace amol
induced he highes concen a ion o enal c ea inine ollowed by indome hacin and aspi in in ha o de . Mix caused
he minimal concen a ion o c ea inine among he exposed a s bu he di e ences among he exposed g oups we e
signi ican .
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Figu e 4 C ea inine concen a ion in he a enal issue
Figu e 5 Succina e dehyd ogenase (SDH) speci ic ac i i y in he a s
The ac i i y o SDH was signi ican ly lowe in all he exposed a s han he Con ol (Fig. 5). Each o he componen d ugs
displayed high SDH ac i i y ela i e o he Mix. Al hough bo h pa ace amol and indome hacin displayed compa ably
high SDH ac i i ies, he di e ence was no s a is ically signi ican .
4. Discussion
Aside in en ional exposu e such as in medica ion, he h ee d ugs can co-exis in en i onmen al media [28, 29, 30] and
in con amina ed ood i ems [31] h ough which humans can be inad e en ly exposed o he mix u es [32]. The s udy
was acco dingly emba ked upon o unde s and he d ug mix u e oxici y and associa ed mechanism in he kidney. A
s udy in gold ish model has demons a ed ha mix u e e ec s a e cha ac e ized by a s ess esponse ha canno be
p edic ed om exposu e o indi idual compounds [33]. Reac i e oxygen species (ROS) a e highly gene a ed in he
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mi ochond ia as a consequence o oxida i e phospho yla ion. Excessi e amoun o he ee adicals a e gene a ed unde
pa hological condi ions, a ec ing edox homeos asis and leading o cellula and issue damage. Supe oxide dismu ase
is a g oup o me alloenzymes ound in all li e o ms. The enzyme o ms he on line o de ence agains ROS-media ed
inju y. The high SOD ac i i y in he con ol g oup sugges ed ha he con ol g oup had a highe capaci y o sca enge
ee adicals and p o ec agains oxida i e damage, which could be a ibu ed o he no mal physiological unc ioning
o SOD in main aining edox balance [34]. The signi ican ly educed supe oxide dismu ase ac i i y in he g oup o a s
ha ecei ed aspi in he e o e indica ed an al e a ion o he edox sys em and an oxida i e impac on he kidney ha
could lead o oxidase s ess. The oxicological e ec o he combined d ugs (Mix) on SOD ac i i y p obably illus a ed
concen a ion addi ion o syne gy; all he componen d ugs we e p esumed o ha e con ibu ed o p oduce a g ea e
e ec han each o he cons i uen d ugs [35]. Lipid pe oxida ion, as indica ed by MDA concen a ion is an impo an
ma ke o oxida i e s ess and cellula damage. Al hough he e a e o he aldehydes esul ing om lipid pe oxida ion
such as hyd oxynonenal, MDA (1, 3-p opane dial) is mo e hyd ophilic and by a highe in concen a ion han o he
lipid-de i ed aldehydes. The odd agains MDA is ha i s concen a ion can be o e -es ima ed because o i s simila
abso p ion maximum wi h o he un ela ed chemical compounds [36]. The signi ican ly highe MDA concen a ion in
he indome hacin g oup compa ed o he pa ace amol g oup sugges s a po en p o-oxidan e ec o indome hacin in
he kidney. The obse ed educ ion o MDA by aspi in could be a ibu ed o i s pu po ed ac i a ion o an ioxidan
de ence mechanism a low dose [37, 38, 39] and migh ha e con ibu ed signi ican ly o he o e all educ ion o e ec
by Mix. Non-p o ein sulphyd yl (NPSH) plays c ucial oles in cellula an ioxida ion and edox homeos asis. NPSH is
con ibu ed p ima ily by educed glu a hione (GSH) and cys eine. Ra kidney is no ed o i s high concen a ion o
cys eine [40]. The e y low concen a ion o NPSH eco ded in Mix g oup ela i e o all o he g oups means enal issues
NPSH was deple ed and he e o e ulne able o oxida i e damage [41] indica ing cumula i e e ec o he cons i uen
d ugs. Succina e dehyd ogenase (SDH), a p o ein assembly in he inne mi ochond ial memb ane is a membe o
ica boxylic acid cycle. The enzyme plays a ole in he educ ion o ubiquinone o ubiquinol in he mi ochond ial
elec on anspo chain. The combina ion o he d ugs p oduced signi ican ly lowe SDH ac i i y. This lowe ing e ec
may ha e esul ed om he cons i uen d ugs inabili y o ac cumula i ely ha would ha e esul ed in addi i e o
syne gis ic e ec s [42, 43]. Acco dingly, he combina ion e ec on SDH is e med an agonis ic [44]. De aul in he
ac i i y o his enzyme will impac nega i ely on cellula ene gy gene a ion and pa hogenesis o ce ain human diseases
such as cance s and neu odegene a ion [45, 46]. C ea inine is a me aboli e o c ea ine phospha e in he muscle and
ollowing i s high wa e solubili y is exc e ed by he kidneys. Se um c ea inine is an impo an ma ke commonly used
o assess muscle mass and kidney unc ion [47]. The obse ed inc ease in he se um c ea inine among he ea ed
g oups could be an indica o o inc eased loss o muscle mass and enal inju y [48]. These esul s may ha e len
c edence o he p e ious epo s associa ing pa ace amol and indome hacin wi h neph o oxici y and enal dys unc ion
[49]. The a e age c ea inine concen a ion in he Mix g oup lowe han he sum o he indi idual d ug e ec s depic ed
an agonis ic e ec and is hough o as he cons i uen d ugs ope a ed a di e en mechanisms o oxici y [44]. Mos
s udies epo ing neph o oxic na u e o he d ug mix u e used e y high doses [50, 51] in sha p con as o his s udy
which used he apeu ic doses o he espec i e d ugs.
5. Conclusion
All he d ugs and hei combina ion displayed a p o-oxidan ac i i y in he enal issues a he apeu ic doses. The p o-
oxidan e ec o he combina ion dose was lowe han he indi idual e ec s.
Compliance wi h e hical s anda ds
Disclosu e o con lic o in e es
The au ho s decla ed no po en ial con lic s o in e es wi h espec o he esea ch, au ho ship, and/o publica ion o
his a icle.
S a emen o e hical app o al
The s udy ecei ed e hical app o al om he LAUTECH E hical Commi ee on use o expe imen al animals.
Funding s a emen
The au ho s ecei ed no inancial suppo o he esea ch, au ho ship, and/o publica ion o his a icle.
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 258-267
265
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