Effect of Prolonged Administration of Artemether-Lumefantrine on Testicular Biomarkers of Oxidative Stress: Ameliorative Effect of Vitamin E

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1 Bsic Sciences of Medicine 2018, 7(1): 1-6 DOI: /j.medicine Effect of Prolonged Administrtion of Artemether-Lumefntrine on Testiculr Biomrkers of Oxidtive Stress: Ameliortive Effect of Vitmin E Onuche A. Dikwo 1,*, Mohmmed U. Kwu 2, Rbiu A. Mgji 1, Ejike D. Eze 3 1 Deprtment of Humn Physiology, Fculty of Medicine, Ahmdu Bello University, Zri, Nigeri 2 Deprtment of Physiology, Fculty of Veterinry Medicine, Ahmdu Bello, University, Zri, Nigeri 3 Deprtment of Physiology, Fculty of Biomedicl Sciences, Kmpl Interntionl University, Ishk-Bushenyi, Ugnd Abstrct Recurrence of mlri infection in Nigeri encourges frequent use of rtemisinin-bsed combintion therpies (ACTs). The ACTs function vi genertion of free rdicls. Rective oxygen species hve been implicted in the etiology of mle infertility. The present study evluted prolonged dministrtion of rtemether-lumefntrine (AL) nd the meliortive effect of vitmin E (VE) on testiculr biomrkers of oxidtive stress. Adult mle Wistr rts (n = 35) were divided into five groups of seven rts ech nd treted twice dily for period of 2 weeks vi orl route s follows; group I: 1ml/kg distilled wter; group II: 1ml/kg olive oil; group III: 2.29 mg/13.74 mg/kg AL; group IV: 2.29 mg/13.74 mg/kg AL mg/kg of VE; group V: 2.29 mg/13.74 mg/kg AL + two weeks recovery (llowed to recover from AL tretment for 2 weeks before smpling). At the end of the experiment, nimls were scrificed nd testes hrvested for nlysis of testiculr biomrkers of oxidtive stress. Testiculr concentrtions of ntioxidnt enzymes glutthione peroxidse (GPx) nd ctlse (CAT) were significntly (P < 0.05) higher in groups IV nd V rts when compred with other groups. Testiculr mlondildehyde (MDA) level ws incresed, while superoxide dismutse (SOD) ws decresed in group III s compred to other groups, though these chnges were not significnt. It is concluded tht, prolonged dministrtion of rtemether-lumefntrine induces significnt ltertions in testiculr ntioxidnt enzymes ctivity suggestive of oxidtive stress nd tht vitmin E nd cesstion of tretment with rtemether-lumefntrine pprecibly meliortes the ltertions in testiculr ntioxidnt enzymes ctivity. Keywords Artemether-lumefntrine, Oxidtive stress, Testes, Vitmin E 1. Introduction Infertility, mjor medicl nd socil predicment, is defined s the inbility to conceive fter t lest, twelve months of regulr nd unprotected intercourse by couples of reproductive ge. It occurs when mn cnnot impregnte proven fertile femle or when womn does not conceive fter one yer of unprotected intercourse with proven fertile mle. In time pst, most of the blme for infertility ws plced on the femle. However, considerble ttention is now being given to the mle fctor. [1, 2]. In Sub-Shrn Afric, the prevlence of infertility is s high s 30% nd the mle contribution in most countries including Nigeri hd been vriously estimted to be between 25 nd 50% [3-5]. Recurrence of mlri (cused by either recrudescence; * Corresponding uthor: onuchedikwo@gmil.com (Onuche A. Dikwo) Published online t Copyright 2018 Scientific & Acdemic Publishing. All Rights Reserved relpse or new infection; [6], hs resulted in the uncontrolled use of rtemisinin-bsed combintion therpies (ACTs) in endemic res of the disese, such s Nigeri. Moreover, self-mediction nd purchse of nti-mlri drugs in the open mrket is rmpnt [7]. Artemisinins trget mlri-infected erythrocytes nd destroy the prsites through genertion of free rdicls [8, 9]. Oxidtive stress medited by rective oxygen species (ROS) is being recognized more commonly s custive fctor in mle infertility [10]. In mles, the developing germ cells, spermtogoni nd spermtocytes of the testis re highly sensitive nd undergo poptosis in conditions tht cuse oxidtive stress [11]. This vulnerbility to oxidtive insult ensues from the high lipid content of the testis. Vitmin E is ft-soluble ntioxidnt nd protects cell membrnes tht re minly composed of ftty cids [12]. The present study evluted prolonged dministrtion of rtemether-lumefntrine (cortem ) nd the meliortive effect of vitmin E (VE) on testiculr biomrkers of oxidtive stress.

2 2 Onuche A. Dikwo et l.: Effect of Prolonged Administrtion of Artemether-Lumefntrine on Testiculr Biomrkers of Oxidtive Stress: Ameliortive Effect of Vitmin E 2. Mterils nd Methods 2.1. Experimentl Animls Thirty-five (35) mle Wistr rts ( g) obtined from the niml house of the Deprtment of Phrmcology nd Therpeutics, Ahmdu Bello University Zri, Nigeri were used for the study. They were kept in rt cges in the Animl House of the Deprtment of Humn Physiology, Ahmdu Bello University, Zri. They hd free ccess to food nd wter nd llowed to cclimtise for period of two weeks before the commencement of the experiment Animls Grouping nd Dosge of Administrtion The thirty-five (n = 35) mle Wistr rts used for the study were divided into five groups of seven (n = 7) rts ech nd treted s follows: Group I: Ech rt ws dministered with distilled wter t 1ml/kg body weight Group II: Ech rt ws dministered with olive oil t 1ml/kg body weight Group III: Ech rt ws dministered with 2.29 mg/13.74 mg/kg Artemether/Lumefntrine dissolved in distilled wter. Group IV: Ech rt ws dministered with 2.29 mg/13.74 mg/kg Artemether/Lumefntrine mg/kg of Vitmin E dissolved in olive oil. Group V: Ech rt ws dministered with 2.29 mg/13.74 mg/kg Artemether/Lumefntrine nd subsequently llowed to recover for 2 weeks during which no tretment ws given. The dose of Artemether/Lumefntrine (Cortem ) used for this study ws clculted from the mnufcturer's recommended dose for mn weighing t lest 35 kg, modifiction of Otuechere et l. [13] Drugs nd Chemicls The Cortem (Artemeter/lumefntrine: 20 mg/120 mg; Novrtis Phrmceuticls Corportion Switzerlnd), Vitmin E (α-tocopherol; Gujrt Liqui Phrmcps Pvt. Ltd. Indi: NAFDAC Reg. No. A4-8322) nd olive oil used for this study were purchsed from reputble phrmceuticl store in Zri, Kdun Stte, Nigeri. The Vitmin E (1000 mg) ws spirted into syringe nd then reconstituted with olive oil (vehicle) prior to dily dministrtion Drug Preprtion nd Administrtion The cortem ws ground to powdered form, mixed with distilled wter nd dministered s n queous suspension. The drug suspension ws continuously gitted during dministrtion in order to deliver the drug homogeneously to the nimls. Vitmin E ws spirted using 1 ml syringe nd reconstituted in olive oil. The respective tretments were dministered to ll rts in ll groups twice dily for period of two weeks using 1.0 ml syringe by orl gvge. However, rts in group V were llowed to recover for 2 weeks fter the lst tretment before they were scrificed Smple Collection At the end of the tretment nd recovery period (group V), ll rts were nesthetised by chloroform inhltion in closed chmber nd therefter, scrificed. The testis ws crefully removed nd homogenised in physiologicl solution (7.4 ph phosphte buffered sline) nd the homogente centrifuged t 1788 g for 10 mins. The superntnt ws removed nd used for ssessment of testiculr biomrkers of oxidtive stress Assy of Testiculr Biomrkers of Oxidtive Stress Assy of biomrkers of oxidtive stress nmely; lipid peroxidtion (mlondildehyde); ntioxidnt enzymes (superoxide dismutse, glutthione peroxidse nd ctlse) ws bsed on the Enzyme-linked immunosorbent ssy (ELISA) technique, using their respective ELISA kits (WKEA MED SUPPLIES CORP). The mnufcturer's mnuls were strictly followed Sttisticl Anlysis SPSS version 17 ws used for the sttisticl nlysis. The dt obtined were clculted by one-wy nlysis of vrince (ANOVA) nd compred using the Tukey's post hoc test. Differences were considered sttisticlly significnt t P < Results 3.1. Effect of Prolonged Administrtion of Artemether-Lumefntrine on Testiculr Mlondildehyde (MDA) Concentrtion The effect of prolonged dministrtion of rtemether-lumefntrine on intr-testiculr mlondildehyde (MDA) concentrtion is shown in figure 1. Testiculr MDA levels were higher in rts treted with AL only (group III) thn in other groups. While, testiculr MDA (µmol/l) concentrtion ws lowest in the AL + Recovery group s follows: AL + Recovery, 1.04 ± 0.10 vs control, 1.61 ± 0.07; OL, 1.08 ± 0.12; AL, 1.44 ± 0.07; AL + Vit E, 1.21 ± 0.10, respectively. However, the differences in MDA concentrtions between the groups were not sttisticlly significnt Effect of Prolonged Administrtion of Artemether-Lumefntrine on Testiculr Concentrtions of Superoxide Dismutse (SOD) The effect of prolonged dministrtion of rtemether-lumefntrine on testiculr concentrtions of SOD is shown in figure 2. Testiculr SOD concentrtion (IU/L) for the five groups were s follows: control, ± 0.09; OL, ± 0.60; AL, 2.26 ± 0.10; AL + Vit E, 2.30 ± 0.08 nd AL + Recovery, 2.29 ± 0.07, respectively. There ws no sttisticl difference in testiculr SOD concentrtion between the groups.

3 Bsic Sciences of Medicine 2018, 7(1): Effect of Prolonged Administrtion of Artemether-Lumefntrine on Testiculr Concentrtions of Glutthione Peroxidse (GPx) The effect of prolonged dministrtion of rtemether-lumefntrine on testiculr concentrtion of glutthione peroxidse (GPx) is presented in figure 3. The testiculr concentrtion of GPx (IU/L) ws significntly (P < 0.05) lower in rts treted with AL only (group III) thn in other groups (AL: ± 1.11 vs control: 44.6 ± 1.44; OL: 45.6 ± 1.03; AL + Vit E: ± 2.63 nd AL + Recovery: 42.4 ± 3.32, respectively; P < 0.05). Mlondildehyde (µmol/l) Tretment Groups Figure 1. Testiculr mlondildehyde level following 14 dys of orl dministrtion of rtemether-lumefntrine, vitmin E nd subsequent withdrwl of the drug for 14 dys. AL= rtemether-lumefntrine; OL = Olive oil; Vit E= Vitmin E Superoxide dismutse (IU/L) Tretment Groups Figure 2. Testiculr superoxide dismutse concentrtion following 14 dys of orl dministrtion of rtemether-lumefntrine, vitmin E nd subsequent withdrwl of the drug for 14 dys. AL= rtemether-lumefntrine; OL = Olive oil; Vit E= Vitmin E

4 4 Onuche A. Dikwo et l.: Effect of Prolonged Administrtion of Artemether-Lumefntrine on Testiculr Biomrkers of Oxidtive Stress: Ameliortive Effect of Vitmin E Glutthione peroxidse (IU/L) b b b b Tretment Groups Figure 3. Testiculr glutthione peroxidse concentrtion following 14 dys of orl dministrtion of rtemether-lumefntrine, vitmin E nd subsequent withdrwl of drug for 14 dys.,b = sttisticlly different; AL = rtemether-lumefntrine; OL = Olive oil; Vit E = Vitmin E b Ctlse (IU/L) Tretment Groups Figure 4. Testiculr ctlse concentrtion following 14 dys of orl dministrtion of rtemether-lumefntrine, vitmin E nd subsequent withdrwl of the drug for 14 dys.,b = sttisticlly different; AL = rtemether-lumefntrine; OL = Olive oil; Vit E = Vitmin E

5 Bsic Sciences of Medicine 2018, 7(1): Effect of Prolonged Administrtion of Artemether-Lumefntrine on Testiculr Ctlse Concentrtions The effect of prolonged dministrtion of rtemether-lumefntrine on testiculr concentrtions of ctlse (CAT) is presented in figure 4. Rts treted with AL only hd significntly (P < 0.05) lower testiculr ctlse (IU/L) concentrtion thn other groups (AL: ± 1.36 vs control: ± 1.24; OL: ± 1.02; AL + Vit E: ± 1.87 nd AL + Recovery: ± 1.17, respectively). 4. Discussion In the present study, rts were treted with 2.29 mg/13.74 mg/kg rtemether/lumefntrine (AL) for two weeks. The choice of dose nd route of dministrtion ws done s recommended for humns, but the 2 weeks durtion of tretment ws to expose the nimls to tretment beyond the recommended durtion, to mimic the extended durtion of tretment ssocited with recurrence of mlri due to incomplete tretment or endemicity. The testis contins n rry of ntioxidnt enzymes such s superoxide dismutse (SOD), ctlse (CAT) nd glutthione peroxidse (GPx). A blnce normlly exists between free rdicls (oxidnts) production nd these ntioxidnt scvenging enzymes ctivities. As result of such blnce, only physiologicl mounts of oxidnts remin, which re needed for the regultion of norml sperm functions, such s sperm cpcittion, hyper-ctivtion, crosome rection nd sperm oocyte fusion. The production of excessive mounts of oxidnts in the testis cn overwhelm the ntioxidnt defence mechnisms nd cuse oxidtive stress, which decreses enzymtic defences of the testes nd cuse dmge to spermtozo thereby compromising sperm qulity nd functions [14]. In the present study, n insignificnt increse in level of mlondildehyde (MDA), mrker of lipid peroxidtion ws observed in the AL group. The reson for this is not fr-fetched, becuse nti-mlril drugs owe their efficcy to their oxidnt effect nd rtemether-lumefntrine in prticulr, genertes free rdicls due to presence of n endoperoxide bridge [15, 16]. This finding grees with n erlier report of incresed MDA concentrtion observed in rtesunte-treted rts [17]. Similrly, significntly lower levels of the ntioxidnt enzymes, glutthione peroxidse (GPx) nd ctlse (CAT), but insignificntly lower concentrtion of superoxide dismutse (SOD) ws observed in the AL group compred with other groups. Superoxide dismutse is considered the first line of defence ginst deleterious effects of oxygen rdicls in cells by ctlyzing the dismuttion of superoxide nion (O 2.- ) to hydrogen peroxide (H 2 O 2 ) nd moleculr oxygen (O 2 ). This ntioxidnt enzyme is present in n essentil mount in the germinl cells of the testis wherein constnt cell division occurs during spermtogenesis [11]. This suggests tht perhps, the testis is highly sensitive to superoxide rdicls (O 2.- ) constntly produced during spermtogenesis. Hence, the SOD immeditely dismuttes O 2.- to yield H 2 O 2 nd moleculr oxygen (O 2 ). The testis now suffers the ill-effect of the eventul genertion of H 2 O 2 which is supposedly cted upon by CAT nd GPx. However, Lysik nd Turner [11], reported tht the level nd ctivities of these enzymes (CAT nd GPx) within the germinl cells re not sufficient enough to quickly buffer the continuously generted H 2 O 2 nd other free rdicls. This could be responsible for the significnt reduction in the levels of these two enzymes in the AL group, s their levels perhps, constntly decline in n ttempt to neutrlise the effect of H 2 O 2. Similr findings of significnt reduction in the levels of ntioxidnt enzymes in the testis of nimls exposed to nti-mlri gents hve been reported [16, 18, 19]. The Vitmin E treted nd recovery groups gve promising results s most of the prmeters investigted in this study were comprble to the bseline levels obtined in the control group. A remrkble but insignificnt decrese in mlondildehyde nd n increse in ntioxidnt enzymes were observed in these groups. Although results in these groups were similr to the bseline levels obtined in the control group, it should be noted tht nimls in control group were not dministered rtemether-lumefntrine. This my suggest tht Vitmin E ssumed its ntioxidnt function, putting the endogenous ntioxidnt enzymes on hold, thereby buffering the negtive effect of free rdicls generted by rtemether-lumefntrine s indicted by the lower level of mlondildehyde in the A/L + Vit E group s compred to the control group. Vitmin E (α-tocopherol) is nturlly occurring ntioxidnt tht plys n importnt role by inctivting hrmful free rdicls produced through norml cellulr ctivity nd from vrious stressors. It termintes lipid peroxidtion nd stbilizes the moleculr composition of cellulr membrnes, thus, preventing the hrmful effects of rective oxygen species (ROS) [20-22]. Restortion to normlcy observed in the recovery group of this study suggests tht the deleterious effects of rtemether-lumefntrine on the prmeters studied could be spontneously corrected with time fter cesstion of dministrtion. 5. Conclusions Bsed on the findings of this study, it is concluded tht prolonged dministrtion of rtemether-lumefntrine in mle Wistr rts induces significnt ltertions in some testiculr ntioxidnt enzymes ctivity, suggesting oxidtive stress, nd, tht vitmin E nd cestion of tretment with rtemether-lumefntrine cuses pprecible meliortion of the ltertions in testiculr biomrkers of oxidtive stress.

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