The antimalarial agent artesunate causes sperm DNA damage and hepatic antioxidant defense in mice

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ARTICLE IN PRESSG ModelUTGEN 402553 1–6

Mutation Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

jo ur nal home page: www.elsev ier .com/ locate /gentoxComm uni t y ad dress : www.elsev ier .com/ locate /mutres

he antimalarial agent artesunate causes sperm DNA damage andepatic antioxidant defense in mice

upriya Singha, Anirudha Girib, Sarbani Giri a,∗

Molecular Cytogenetics Laboratory, Department of Life Science and Bioinformatics, Assam University, Silchar 788 011, IndiaEnvironmental Toxicology Laboratory, Department of Life Science and Bioinformatics, Assam University, Silchar 788 011, India

r t i c l e i n f o

rticle history:eceived 31 August 2014eceived in revised form 4 November 2014ccepted 7 November 2014vailable online xxx

eywords:rtesunateomet assay

a b s t r a c t

Artesunate is an artemisinin derivative effective against multidrug resistant malaria. We analyzed theeffects of artesunate 40 mg/kg b.w. as a single dose (ART1) or 13.3 mg/kg b.w. for 3 days at 24 h intervals(ART2) on mice spermatozoa at morphological and molecular level, and hepatic antioxidant status fol-lowing 24 h and 35 days following exposures in vivo. Artesunate significantly reduced epididymal spermcount and increased the frequency of sperms with abnormal head morphology following 24 h of exposure.Comet assay analysis revealed significant increase in DNA strand breaks in spermatozoa evidenced byabout 3-fold increase in comet tail DNA and up to 10-fold increase in Olive tail moment following 35 daysof artesunate treatment. The damage index was significantly higher in the treated groups (40.27 ± 6.62

NA damage index, Lipid peroxidationeduced glutathioneuperoxide dismutase

and 37.07 ± 5.35 for ART1 and ART2 respectively) as compared to the control group (16.13 ± 3.21) indi-cating the genotoxic effect of artesunate. The significant reduction in GSH, SOD and increase in lipidperoxidation indicate involvement of oxidative mechanisms in artesunate induced toxicity in mice. Thepresent study suggests that artesunate has the potential to breach the testis–blood barrier and causetoxicity to male germ cells which may have implications in male reproductive toxicity.

© 2014 Published by Elsevier B.V.

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. Introduction

Malaria still remains one of the major diseases worldwideausing deaths of millions of people every year. Among theotal reported cases, 80% are reported from developing countries.regnant woman and children under five years of age are theajor victims [1]. The widespread emergence of resistant malaria

arasites against antimalarial drugs has lead to the quest for alter-ative treatments. Artemisinin or qinghaosu is an important classf anti malarial drugs isolated in 1972 from the leaves of Artemisiannua, a Chinese herb used for the treatment of fever for cen-uries [2]. Artemisinin and its derivatives are known to exhibitotency against Plasmodium falciparum and Plasmodium vivax [3].mong the compounds in the artemisinin group, artesunate (ART)

Please cite this article in press as: S. Singh, et al., The antimalarial agendefense in mice, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014

nd artemether are most widely used [4]. Transmission of malariaepends on the geographical and climatic condition, immuno-

ogical status of the exposed population as well as availabilityf mosquito species. Poor literacy rate, lack of awareness, less

∗ Corresponding author at: Department of Life Science & Bioinformatics, Assamniversity, Silchar 788 011, India. Tel.: +91 3842 240400.

E-mail address: [email protected] (S. Giri).

ttp://dx.doi.org/10.1016/j.mrgentox.2014.11.001383-5718/© 2014 Published by Elsevier B.V.

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stringent laws, drug availability without prescription, frequentinfections by malaria parasite, make people prone to indiscriminateconsumption of antimalarial drugs. The mechanism of action of ARTinvolves the heme-mediated decomposition of the endoperoxidebond to produce free radicals which kill the parasite accumulatedin the erythrocytes [5]. The widespread use of ART has made itimperative to characterize its potential toxicity, especially thoserelated to the germ cells.

Reproductive toxicity has been associated with other antimalar-ial drugs; for instance chloroquine has been reported to inducesperm head abnormality in vivo studies [6]. Raji et al. [7] showedthat artemether, leads to a significant reduction in the spermmotility, viability, sperm count and serum testosterone levels in adose-dependent manner in rats. ART has also been shown to causea decrease in sperm motility in guinea pigs [8]. ART induces toxic-ity in female reproductive system. It was shown to reduce serumprogesterone concentration, degenerate the deciduas in fetus oftreated pregnant rats [9]. ART causes embryo-fetal toxicity causingembryo deaths and malformations [10,11]. Toxicity reports on ratsindicate that under certain conditions, ART suppresses spermato-

t artesunate causes sperm DNA damage and hepatic antioxidant), http://dx.doi.org/10.1016/j.mrgentox.2014.11.001

genesis in testis [12]. Therefore, it is essential to evaluate the effectof ART on spermatogenesis and sperm DNA integrity. Sperm headmorphology assay (SHA) is an inexpensive, sensitive and reliablemethod to evaluate germ cell abnormalities and is widely used to

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dx.doi.org/10.1016/j.mrgentox.2014.11.001


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ARTICLEUTGEN 402553 1–6

S. Singh et al. / Mutation

dentify germ cell mutagens and thus repeatedly used as indicatorf toxicity and mutagenicity in mammals [13–17].

Liver is the primary organ for xenobiotic metabolism in the body.rugs are metabolized and converted to more polar compounds in

iver to facilitate excretion. Therefore, liver is highly susceptibleo drug induced toxicity. Like other drugs ART is also metabolizedn hepatocytes and since the drug is known to be associated withxidative stress [5], it is important to investigate drug inducedxidative stress in liver cells. Comet assay is an effective, sensi-ive and rapid method to evaluate DNA damage. Thus the presenttudy was aimed at evaluating the effect of ART on male germinalells, study the extent of oxidative stress induced by it as well asvaluate DNA damage in mice test system.

. Materials and methods

.1. Test chemicals

Artesunate (3R,5aS,6R,8aS,9R,10S,12R,12aR)-decahydro-3,6,9-trimethyl-3,12-poxy-12H-pyrano(4,3-j)-1, 2-benzodioxepin-10-olhydrogen succinate), CAS Reg-stry No: 83507-69, was obtained from Oscar remedies, India. Mitomycin C

as purchased from Biochem Pharmaceuticals Industries Ltd. (Daman, India).-Thiobarbituric acid and Triton X were purchased from Hi-Media laboratoriesMumbai, India). Tris buffer, Pyrogallol, DTNB, normal melting point (NMP) agarose,ow melting point (LMP) agarose, Tris buffer, ethidium bromide (EtBr) and Tris HCl

ere procured from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). All otherhemicals used were of analytical grade. The buffer for stains and reagent solutionsere always prepared in glass-distilled water. The chemicals to be administered to

he mice were prepared in physiological saline.

.2. Animals and dose

The study was approved by the Institutional Ethical Committee of Assam Uni-ersity. Swiss albino mice (10–12 weeks; 20–25 g) were used in the study. Thenimals were housed in laboratory conditions at room temperature of 25 ± 5 ◦C inhotoperiod-controlled environment (12L:12D cycles). These were provided withood pellets (Amrut Laboratory Animal Feeds, New Delhi) and water ad libitum. Alto-ether 75 male mice were used for study were divided into 4 different treatmentroups. The treatment groups consisted (i) vehicle control (normal saline), (ii) posi-ive control (2 mg/kg b.w. of mitomycin C), (iii) ART1 (40 mg/kg b.w. of ART given as

single acute dose) and (iv) ART2 (13.4 mg/kg b.w. per day given for 3 days at 24 hntervals). The dose of ART used in the present study is lower than the body surfacerea normalized human equivalent dose [18,19]. Sperm head abnormality assay wasarried out at 24 h and 35 days following the treatments. Five animals from each ofhe 4 treatment groups were used for analysis at each of the 2 time points studied.ipid peroxidation (LPO), superoxide dismutase (SOD) and GSH levels were mea-ured after 24 h of the treatments for which 5 animals from each of the 4 treatmentroups were used. DNA damage analysis using comet assay was done 35 days afterhe treatments. The 3 treatment groups namely control, ART1 and ART2 consistedf 5 animals each.

.3. Sperm head abnormality assay

The animals were sacrificed by cervical dislocation. Both the cauda epididymisere dissected out and placed in normal saline. The sperms were released byechanical disruption and washing of the epididymis. A drop of suspension was

meared on clean glass slide, air dried and fixed in methanol for 10 min. The slidesere stained in Eosin Y and 1000 sperms were scored per animal and the abnormal-

ties were categorized as close as described by Wyrobek and Bruce [17]. Epididymalperm count was made using Neubauer’s hemocytometer.

.4. Comet assay

The DNA damage studies were carried out using comet assay according to theethod of Singh et al. [20] with modifications of Tice et al. [21]. Animals were

acrificed by cervical dislocation after 35 days of the treatments. Both cauda epi-idymises were dissected out and sperms were released into 1 ml of PBS pH 7.4sing a watchmaker’s forceps. The sperm count was done using a Neubauer cytome-er. The sperm suspension (at the concentration of 1 × 106 cells/ml) was mixed 1:10ith 250 �l molten LMP agarose, and samples of 85 �l of the mixture were rapidly

pread on comet slides. Slides were prepared in triplicate. Slides were immersedn cold lysis solution at pH 10 for 60 min in 4 ◦C. The lysis solution consisted of


.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma base, 1% Triton X100 and 10% DMSO.fter lysis the DNA was allowed to unwind in the electrophoresis buffer (300 mMaOH:1 mM Na2EDTA at pH 13.5) for 20 min. This was followed by electrophoresisonducted at a constant voltage of 24 V and 300 mA at 4 ◦C. Slides were neutralizedn 0.4 M Tris buffer (pH 7.5) for 5 min and finally rinsed in distilled water. The slides

PRESSrch xxx (2014) xxx–xxx

were stained with EtBr (20 �g/l) and rinsed in distilled water to wash off excess stain.Slides were scored using image analysis system (Kinetic imaging; Andor Technology,Nottingham, UK) attached to a fluorescence microscope (Leica, Wetzlar, Germany)equipped with appropriate filters. Cells at 100× magnification were analyzed usingKomet 5.5 software. Images of 150 cells per animal were analyzed (50 cells per slide)to record various comet parameters like head DNA, tail DNA, tail length and olivetail moment etc.

We calculated the damage index (DI) for each treatment group by assigning adamage score to a classified data set based on comet tail length. The differentiallydamaged cells were classified into 5 classes as reported earlier [22] except for the factthat our classification is based on objective measurement of the tail length using theComet software. The damage classes 0, 1, 2, 3, and 4 had tail lengths 0, >0–5, >5–10,>10–20 and >20 �m respectively. Therefore, damages were evaluated in a scale of 0(i.e. 0 × 100 indicating completely undamaged cells) and 400 (i.e. 4 × 100 indicatingall cells are highly damaged with tail length > 20 �m). The DI was calculated usingthe following formula:

DI = 15

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Xn

where ‘X’ is the class number and ‘n’ is the percentage of cells in each class.

2.5. Biochemical estimations

Lipid peroxidation in the liver was measured by the thiobarbituric acid-reactingsubstance (TBARS) and was expressed in terms of malonaldehyde (MDA) content[23]. Tissue homogenate (10%) was prepared in chilled normal saline and centrifugedat 3000 rpm for 10 min. One milliliter of the supernatant was incubated at 37 ◦C for2 h. Then, 1 ml of trichloroacetic acid (10%) was added, mixed properly and cen-trifuged at 2000 rpm for 5 min (4 ◦C). Equal volumes of thiobarbituric acid (0.67%)was added to the 1 ml of supernatant and mixed thoroughly followed by incubationin boiling water bath for 10 min. The samples were cooled, diluted and O.D. wasmeasured at 535 nm on Genesys-20 spectrophotometer (Thermo Scientific, USA).Results were expressed as nmol MDA/g wet tissue.

The method described by Marklund and Marklund [24] was used for the assay ofSOD activity. The liver homogenate (10%) was prepared and pyrogallol (25 mM) wasused for SOD activity estimation. Absorbance was taken at 420 nm and results wereexpressed in terms of (U) per gram of wet tissue. Estimation of cellular levels of GSHwas done using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) method [25]. Tissue washomogenized in 5–8 ml of EDTA (0.02 M), diluted with ice cold water and 1 ml oftrichloroacetic acid (50%) was added to it followed by addition of Tris buffer (0.4 M;pH-8.9) and (0.01 M) DTNB. Absorbance was measured at 412 nm and results wereexpressed as mmol/g tissue.

2.6. Statistical analysis

Quantitative data were expressed as mean ± SE and analyzed by one way analy-sis of variances (ANOVA) followed by Tukey’s multiple comparison test for pair wisecomparison between study groups. Prior to analysis, the data were tested for nor-mality in distribution and were square root transformed wherever required prior toanalysis. Differences were considered statistically significant when P < 0.05.

3. Results

3.1. Changes in sperm count and sperm head abnormality

The findings of the sperm head abnormality assay are presentedin Table 1. It has been observed that the percentage of abnormalsperms increased after ART treatment for 24 h and 35 days. Signif-icant decrease in epididymal sperm count (P < 0.05) was observedonly in the experimental group ART1 as compared to the controlgroup at both 24 h and 35 days of the treatments (Table 1). In thechronic treatment group (ART2) though reduced sperm count wasrecorded, these were not statistically significant.

In sperm head abnormality assay, both the experimental groupsART1 and ART2 showed statistically significant (P < 0.001) increasein the frequency of sperms with abnormal head morphology com-pared to control group after 24 h as well as 35 days (Table 1).


Aberrant head morphology like amorphous, beaked, hooked, hookless, altered head, triangular, banana, pinheaded, double-headedetc. were observed (Table 2). Among all types of abnormalities,amorphous form was more prevalent than the other types.

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Please cite this article in press as: S. Singh, et al., The antimalarial agent artesunate causes sperm DNA damage and hepatic antioxidantdefense in mice, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.11.001

ARTICLE IN PRESSG ModelMUTGEN 402553 1–6

S. Singh et al. / Mutation Research xxx (2014) xxx–xxx 3

Table 1Incidence of sperm head abnormality in mice treated with artesunate for 24 h and 35 days.

Dose and treatment 24 h 35 days

Treatment Route No. of spermstudied (n)

No. of abnormalsperm

% of abnormalsperm(mean ± S.E.)

Total sperm count/epididymis ×106

(mean ± S.E.)

No. of abnormalsperm

% of abnormalsperm(mean ± S.E.)

Total sperm count/epididymis ×106

(mean ± S.E.)

CON i.p. 5000/5 164 3.3 ± 1.0 30.0 ± 1.2 151 3.0 ± 0.1 32.6 ± 1.5MMC i.p. 5000/5 454 9.1 ± 1.3 16.6 ± 0.7 472 9.4 ± 0.2 22.3 ± 1.0ART1 i.p. 5000/5 360 7.2 ± 0.1** 21.6 ± 1.8*,a 400 8.0 ± 0.2** 26.3 ± 0.3*

ART2 i.p. 5000/5 312 6.3 ± 0.3** 28.0 ± 1.3a 340 7.3 ± 0.2** 29.3 ± 0.5

Control: only normal saline was given; MMC: mitomycin C was used as positive control; ART1: artesunate (40 mg/kg b.w.); ART2: artesunate (13.4 mg/kg × 3 days); n: numberof animals. Statistical analysis: Students’ t-test.

* Significantly different from control: P < 0.05.** Significantly different from control: P < 0.001.a P < 0.05. Values having similar superscripts are significantly different from each other.

Table 2Distribution of sperms with abnormal head morphology into different aberration types.

Treatment Total spermsstudied (n)

Fixation time(h/d)

Total aberrantsperms

Types of abnormalities studied

Banana Amorphous Beaked Hooked Hook less Pin-head Altered head Triangular

CON 5000/5 24 h 164 31 79 16 8 6 1 4 19MMC 5000/5 24 h 454 58 229 35 25 21 6 48 32ART1 5000/5 24 h 360 32 225 31 16 22 1 20 13ART2 5000/5 24 h 312 48 162 27 17 14 0 26 18CON 5000/5 35 d 151 28 74 11 4 10 0 14 10MMC 5000/5 35 d 472 70 163 63 29 40 11 50 46ART1 5000/5 35 d 400 42 222 36 15 28 2 29 26ART2 5000/5 35 d 340 40 212 28 8 18 0 22 12

Control: only normal saline was given; MMC: mitomycin C; ART: artesunate. ART1: artesunate (40 mg/kg b.w.); ART2: artesunate (13.4 mg/kg × 3 days); n: number of animals.

Fig. 1. Photomicrographs of comet DNA and histograms showing changes in comet parameters as well as damage index following artesunate treatment. The photomicrographsshow a control nuclei, comet DNA with varying amounts of tail DNA and a hedgehog nuclei without clear distinction of head and tail. Artesunate 40 mg/kg b.w. (ART1) and13.5 mg/kg b.w. × 3 days (ART2). Significantly different from control at P < 0.01 (*) and P < 0.001 (**).


ARTICLE IN PRESSG ModelMUTGEN 402553 1–6

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Fig. 3. Histograms showing changes in lipid peroxidation (MDA level), GSH andSOD in hepatic tissue following 24 h of artesunate treatment. CON: vehicle control;

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ig. 2. Histogram showing distribution of cells in different damage classes. C0–C4epresent different damage classes as defined in the methodology section. Arte-unate 40 mg/kg b.w. (ART1) and 13.5 mg/kg b.w. × 3 days (ART2).

.2. Changes in sperm comet assay parameters

Comet assay, which detects DNA single strand breaks, is onef the contemporary techniques widely applied in genetic toxico-ogy. In the present study, ART given either as a single dose or asractionated doses for three days at 24 h intervals caused signifi-ant damage to DNA as observed under the microscope. Besides,ccasional ‘Hedgehog cells’ characterized by the absence of clearifferentiation into head and tail regions were also observed (photoanel in Fig. 1). Quantitative estimate showed significant increase

n the % of tail DNA (P < 0.001) in the ART treated groups comparedo the control group (Fig. 1). The tail DNA was up to 3.15 fold highern the treated groups than the control. The olive tail moment wasigher in the treated groups between 8 and 10 folds (Fig. 1) givingn indication of the nature and extent of the damage. The DI wasignificantly (P < 0.001) higher in the treated groups (40.27 ± 6.62nd 37.07 ± 5.35 for ART1 and ART2 respectively) as compared tohe untreated control (16.13 ± 3.21). Notably, no significant differ-nce in the comet parameters could be found between the groupsiven 40 mg/kg b.w. as a single bolus dose or as fractionated dosesFig. 1). The distribution of cells in different damage classes (Fig. 2)learly shows that in the control group majority of the cells weren the damage class 0 and 1 (tail length between 0 and 5 �m)

hereas in the ART treated groups cells in the damage class 4 (tailength > 20 �m) were more frequent.

.3. Changes in lipid peroxidation levels

Lipid peroxidation data (Fig. 3) shows that both doses of ART sig-ificantly increased MDA level in the hepatic tissue after 24 h of thereatments. ART1 and ART2 showed increased MDA levels whichere 13.3 ± 0.7 and 8.0 ± 0.2 nmol MDA/g wet tissue respectively as

ompared to the control value of 3.5 ± 1.1 nmol MDA/g wet tissue.he increase was statistically significant in ART1 group (P < 0.01)nd in ART2 (P < 0.05) when compared to the control group (Fig. 3).hen compared between the two tested doses, the single dose

ART1) produced more MDA than the fractionated dose (ART2)hich however, was not statistically significant.

.4. Changes in reduced glutathione levels


Compared to the control group (9.0 ± 0.5 �Mol/g wet tissue)he hepatic levels of GSH significantly decreased (P < 0.001) in ARTreated groups. In ART1 it was 3.0 ± 0.1 mmol/g whereas in ART2he value recorded was 4 ± 0.2 mmol/g of tissue (Fig. 3).

MMC: mitomycin C (2 mg/kg b.w.) as positive control; Artesunate 40 mg/kg b.w.(ART1) and 13.5 mg/kg b.w. × 3 days (ART2). Significantly different from control atP < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

3.5. Changes in SOD activity

Hepatic SOD activity was found to be significantly lowered inboth the tested doses after 24 h of ART treatment. SOD activity was2.3 ± 0.5 U (P < 0.001) and 3.1 ± 0.5 U (P < 0.001) in ART1 and ART 2groups respectively as compared to the control group in which theactivity was 8.0 ± 0.3 U (Fig. 3). However, no significant differencein SOD level was found between the single and the fragmenteddoses of ART.

4. Discussion

The present study indicate that ART treatment produce toxiceffects in the male germ cells in mice and clarifies the genotoxicpotential of ART. A significant reduction in epididymal sperm count,


increase in the frequency of sperms with abnormal head morphol-ogy and induction of oxidative stress were among the short-termeffects of ART exposure. Induction of sperm head abnormality hasbeen reported to be associated with genetic damage such as point

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utation [26], alterations in testicular DNA [27] or interference ofhemicals with the genetically controlled differentiation process ofhe spermatogonial cells [28,29]. It is important to note that spermsre terminally differentiated cells and do not undergo furtheritotic division. Although some recent studies have found evi-

ence of post-meiotic transcription during spermatogenesis [30],resently there is no evidence to suggest transcriptional activity inpermatozoa [31]. Therefore, the higher incidence of sperms withbnormal head morphology observed at 24 h may not be due to theenotoxic effect, if any, caused by ART.

The mechanism of antimalarial activity of ART involves gener-tion of free radicals [5,32,33] and reactive oxygen species (ROS)ill parasites by damaging their cell membrane. In the presenttudy ART induced an increase in MDA levels suggesting the accu-ulation of lipid peroxides formed due to the interaction of ROSith membrane lipids (Fig. 3). In addition, the significant decrease

n GSH and SOD level in hepatocytes following ART treatmentuggest a reduction in the free radical scavenging ability of theffected cells. Spermatozoa are particularly sensitive to oxidativetress due to lack of abundant cytoplasmic space to accommo-ate antioxidant enzymes that offer first line of defence againstree radical attack as well as availability of abundant targets suchs DNA and polyunsaturated fatty acids for peroxidative dam-ge [34,35]. Polyunsaturated fatty acids in the plasma membranelay an important role in membrane structure and function [36].herefore, the abnormal head morphology observed at 24 h ofRT treatment is the likely consequence of plasma membraneamage due to lipid peroxidation. Oxidative stress induces abnor-al sperm head morphology by damaging membrane lipids [37].xidative stress is known to cause DNA damage and has been

eported to accelerate apoptosis, which may have implicationsn male fertility [38,39]. The significant decrease in sperm countt 24 h following ART1 treatment observed in the present studyTable 1) could be due higher rate of cell death induced by apopto-is. Oxidative stress has been reported to reduce the sperm countn epididymis and increase the percentage of defective sperms inats [40].

Oxidative stress can have long-term impact on male germ cellifferentiation. It has been reported that oxidative stress inhibitspermatogenesis, decreases SOD, and induces DNA damage as wells apoptosis [41]. Germ cells take about 35 days for differentiationnto functional sperms. In the present study, unlike in the 24 h expo-ure period, occurrence of significantly higher frequency of spermsith abnormal head morphology indicates the genotoxic effect ofRT (Table 1). The present findings are consistent with other stud-

es reporting induction of sperm head abnormality in mice exposedo genotoxic agents [6,42].

Although all cellular macromolecules are subject to damage byeactive oxygen species, the primary deleterious consequences ofxidative stress probably arise from damage to DNA [43]). Cometssay in sperm cells 35 days following ART treatment significantlyncreased all the measured DNA damage parameters including theI (Fig. 1) indicating the genotoxic potential of ART. The dam-ge distribution pattern (Fig. 2) clearly showed that in the controlroup virtually all the cells showed either no damage to slightamage (damage class C0–C1), whereas most of the ART treatedells were highly damaged (damage class C4). In the present studye measured ‘olive tail moment’ [44] defined as the product of

he tail DNA% and the tail length instead of ‘comet moment’ [45]hich is the length of the comet without any distinction of theead and tail regions. The olive tail moment often provides a mea-ure of both the nature and extent of damage although the comet


oment is easy to measure and useful over a larger dose range. Inddition, differentiation of the comet into separate head and tailegions allows to selectively eliminate the ‘Hedgehog’ and ‘ghost’ype of comets (where the entire comet is recognized as the head)

PRESSrch xxx (2014) xxx–xxx 5

and hence gives a more accurate measure of DNA strand breaks.The anticancer drug methotrexate induced strand breaks are accu-mulated in the replicating DNA of spermatogonial cells which theauthors suggest to be the consequence of depleted nucleotide pooland/or the impairment of the repair mechanisms [46]. It is possiblethat similar mechanism plays a role in ART induced genotoxicity.Therefore, the sperm head abnormalities observed following 35days of ART treatment may be the consequence of an impaireddifferentiation process of the spermatogonial cells due to geno-toxic effect of ART. It has been reported that long-term treatmentof low doses of ART induced visible lesions in the testicular andepididymal tissues of rats in vivo. Further, cultured Sertoli cellsexposed to ART show dose and time dependent changes in cellviability and ds-DNA integrity [47]. ART has also been reportedto induce dose-dependent increase in DNA damage and apopto-sis in cultured HepG2 [48] and LN-229 human glioblastoma cells[49].

Liver is the primary organ for drug metabolism. ART is apro-drug, which is readily converted to artenimol (the active anti-malarial agent) by cytochrome P-450 (CYP2A6) enzyme in the liver[50] during which free radicals are generated [32]. In the presentstudy, the increased hepatic MDA levels, in ART treated groups issuggestive of peroxidative damage to membrane lipids by free radi-cals. Under normal circumstances, the antioxidant enzymes of thecell get activated and effectively scavenge the free radicals restor-ing the normal cell environment. However, ART is known to inhibitprotein synthesis in most mammalian systems [51]. Therefore, thesignificant decrease in GSH and SOD observed following ART treat-ment could result from enhanced utilization without augmentedproduction. ART has been reported to be a substrate of the glu-tathione detoxification system [52].

The present findings suggest that disruption of sperm morphol-ogy and the concomitant presence of high levels of DNA damagein the sperm nucleus could involve a common mechanism in theform of oxidative stress induced by ART. It has been reported thatthe presence of antioxidant mechanisms (especially the expressionof the antioxidant enzymes SOD, GPX and catalase) are importantduring the spermatozoid voyage to achieve fertilization [53]. Thealteration of sperm head morphology caused by ART due to mem-brane damage, because of oxidative stress or due to DNA damageat the spermatogonial stage, could perturb their capacity for fertil-ization and may have implications in male fertility.

5. Conclusion

The antimalarial drug ART, which is found to be effectiveagainst multidrug resistant malaria, has the potential to breach thetestis–blood barrier and cause toxicity to male germ cells whichmay have implications in male reproductive toxicity. The primarymechanism involved could be oxidative damage to membranelipids and DNA. Therefore, self-medication or unrestricted use ofART should be avoided.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgement

The financial support by the Department of Science and Tech-


nology, Government of India under DST-FIST program to theDepartment of Life Science and Bioinformatics, and fellowship toSS from University Grants Commission, Government of India arethankfully acknowledged.

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The antimalarial agent artesunate causes sperm DNA damage and hepatic antioxidant defense in mice