SUB-ACUTE CHLOROQUINE TOXICITY ON TESTIS OF SWISS ALBINO MICE AND ITS AMELIORATION BY CURCUMIN

SUB-ACUTE CHLOROQUINE TOXICITY ON TESTIS OF SWISS ALBINO MICE AND ITS AMELIORATION BY CURCUMIN

Ketaki R. Desai, Pragnesh B. Patel and Zeba Siddiqui and Hyacinth N. Highland ^*

Department of Zoology, BMT & HG, School of Sciences, Gujarat University, Navrangpura, Ahmedabad - 380009, Gujarat, India.

ABSTRACT: The objective of the present study is to investigate the adverse effect of chloroquine (CQ) toxicity on reproductive tissues and its attenuation by curcumin. Chloroquine (CQ) has been used widely as an antimalarial drug due to its quick action on blood schizontocide of different malarial parasite species for prophylaxis and treatment of malaria. Thirty-six male mice were divided into six experimental groups: control, curcumin (80 mg/kg b.w.), CQ low dose (100 mg/kg b.w.), CQ mid dose (200 mg/kg b.w.), CQ high dose (300 mg/kg b.w.) and CQ high dose + curcumin treated group. An oral dose of artesunate and curcumin was administered for a period of 14 and 21 days to male Swiss albino mice. Results obtained indicated significant alterations in biochemical parameters as well as energy metabolism parameters in testicular tissue of animals after the treatment with chloroquine. Moreover, sperm count, sperm motility percent, sperm viability and sperm morphology of cauda epididymis of chloroquine treated mice revealed significant fluctuations as compared to the control group. Administration of curcumin along with the chloroquine revealed comparable values of biochemical as well as sperm parameters with the control group. Hence, obtained results clearly indicates the strong mitigative potential of curcumin against chloroquine induced toxicity in Swiss albino male mice.

Chloroquine, Curcumin, Antimalarial drug, Biochemical, Testicular

INTRODUCTION: Globally the major public health issue in endemic regions ¹ causing high mortality and morbidity, is the vector borne disease Malaria. The disease is a multifactorial burden - economically, medically, and a health calamity as the synergy of co-infections with the HIV and Tuberculosis is high ². About 90% cases of malaria occur in sub-Saharan Africa where the disease contributes substantially to underdevelopment and places a severe strain on limited health care facilities ³.

The treatment of malaria has posed great challenge to medicine and development of efficient antimalarial drugs. This is due to the development of resistance of parasite to mainly antimalarial agents ^{4, 5} resulting in huge impact on the socio-economy ⁶. The possibility of overdose and misappropriation in the usage of antimalarial agents are very common, all of which could lead to toxic effects of the drugs ^{7, 8}.

Chloroquine (CQ) has been used for the treatment of diverse diseases. It is an aminoquinoline commonly used in the tropics to treat malaria ⁹. When unprotonated, it can diffuse across cell membranes, get protonated and accumulate in acidic organelles such as the lysosomes. This lysosomotropic property results in the inhibition of lysosomal enzymes and makes it useful in the treatment of the malaria parasite ¹⁰. Given the lack of affordable alternatives, chloroquine remains the first-line antimalarial agent in most African countries. In recent decades chloroquine has been used so extensively that it has now lost its efficacy against malarial parasites. Indeed, resistance to chloroquine is so widespread that this drug has been rendered useless in some parts of the world, especially South East Asia ¹¹.

Chloroquine is a lysomotropic agent which inhibits the degradation of internalized human chorionic gonadotropin (hCG) in Leydig cell ¹² and luteal cells ¹³. It has been shown to have varied effects on male reproductive functions including fertility reduction in male rats ¹⁴. Chloroquine was shown to inhibit testosterone secretion in hCG-stimulated testis of pubertal rats ⁹. The evaluation of antimalarial drugs for possible reproductive toxicity especially at doses higher than recommended doses becomes very important as both malaria and infertility are of global concern.

Although there have been few documentaries, review of literature reveals a paucity in the field of reproductive toxicity of the said drug. Antimalarial drugs have been implicated in male infertility and CQ has been found to have dose dependent reduction in fertility of male rats ^{15, 16}.

The global use of this antimalarial drug thus made its detailed investigation on male fertility imperative. Further, the literature documents a couple of chloroquine induced reproductive and other toxicities, though, the awareness regarding the same is scarce. This necessitates the need of mitigation of toxicity.

Medicinal plants and natural herbal products have potential antioxidant activity and are therefore often administered along with chemotherapeutic agents to provide better protection against their toxic side effects ¹⁷. Natural products have a wealth of applications. Some of them are used as drugs, while others possess important biological properties or are used as dietary supplements, as dyes, flavouring agents, or ingredients in the cosmetics industry ¹⁸.

One such medicinal plant is Turmeric (Curcuma longa Linn. Syn C. domestica Valeton) which is extensively used as a spice, food preservative and colouring material commonly used in the Indian subcontinent ^{19, 20}. Since the time of Ayurveda (1900 BC) numerous activities have been assigned to turmeric for a wide variety of diseases and conditions, including those of the skin, pulmonary, and gastrointestinal systems, aches, pains, wounds, and liver disorders ¹⁹.

Various phytochemicals are present in turmeric and one of them is ‘Curcumin’ also known as diferuloyl methane and exhibits antioxidant, anti-inflammatory, anti-mutagenic, anti-carcinogenic, membrane-stabilizing, and hypolipidemic properties which constitute an important part of its therapeutic effects ^{21 - 23}. Curcumin has also been shown to retard lipid peroxidation and ameliorate chemically induced oxidative stress ^{24, 25}. The high oxidative potential of curcumin has not been extensively explored as specially in curbing reproductive toxicity in mice. Thus the present investigation was focused on the mitigating potential of curcumin on reproductive toxicity in male Swiss mice.

The study was hence designed to explore the possible protective effect of curcumin on chloroquine induced reproductive damage in albino mice.

MATERIALS AND METHODS:

Animals and Chemicals: Healthy adult male albino mice, Mus musculus of a Swiss strain, weighing between 25 and 40 g, were obtained from Cadila Pharmaceuticals (Dholka, India). All the animals were acclimatized 7 days before commencement of treatment and were maintained under controlled conditions, with 12 h light-dark cycles at a temperature of 26 ± 2°C and relative humidity of 30 - 70%. Animals were randomized into control and experimental groups and were caged separately. Standard chow (obtained from Amrut Laboratory, Baroda, India) and water were given ad libitum. Experiments were conducted in accord with the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India, and experimental protocols were approved by the institutional animals’ ethics committee, under registration no. 167/1999/ CPCSEA. Chloroquine phosphate (99.30% pure) was generously gifted from IPCA Laboratories Ltd. (Mumbai, India). Curcumin and other chemicals were procured from Himedia Laboratories (Mumbai, India) and Sigma-Aldrich (Dorset, UK). All chemicals used in the experiment were of analytical grade.

Experimental Design: A stock solution of CQ was prepared in double-distilled water and orally given to mice via feeding canula with a hypodermic syringe. All doses for CQ were derived from its oral LD₅₀ value (500 mg/kg) ²⁶. The dose for curcumin is based on an earlier work ²⁷. Animals were divided into the following six groups: group 1, control (given distilled water only); group 2 was given 80 mg/kg b.w. of curcumin; group 3, 100 mg of CQ/kg b.w.; group 4,200 mg CQ/kg b.w.; group 5,300 mg CQ/kg b.w.; group 6, high dosage of CQ+curcumin (300 mg CQ/kg b.w. + 80 mg/kg b.w.) and treatment for 14 and 21 days.

TABLE 1: EXPERIMENTAL DESIGN

 All the groups were treated for a 14 and 21 days period. At the end of each treatment, animals were weighed and sacrificed using light ether anaesthesia. Testes were taken for biochemical and histological evaluation. Animals were then euthanized via exsanguination.

Tissue Collection: After termination of the treatment period, animals were euthanized and dissected. Testes were dissected out carefully and weighed. Tissue was then processed and homogenate was prepared according to standard biochemical protocols.

Protein Estimation: Protein estimation was done by using the standard protocol of Lowry et al., (1951) ²⁸. The optical density (OD) of blue colour was read at 540 nm in systronics digital spectrophotometer 167 against blank.

Acid Phosphatase (ACPase) Activity: Activity of ACPase was determined by the method of Bessey et al., (1946) ²⁹. ACPase catalyzes hydrolysis of p-nitrophenol nitrate at pH 4.8, liberating paranitrophenol and inorganic phosphate. The liberated p-nitrophenol combines with NaOH to form a yellow colored complex which is measured at 420 nm and is directly proportional to the enzyme activity. Enzyme activity was expressed as µ moles of p-nitrophenol released/30 minutes/mg protein.

Alkaline Phosphatase (ALKPase) Activity: Alkaline Phosphatase (ALPase) activity was determined by the method of Bessey et al., (1946) ²⁹. The enzyme ALPase hydrolyses the substrate p-nitrophenyl phosphate into inorganic phosphate and p-nitrophenol. The quantity of p-nitrophenol released under standardised condition was measured at 410 nm. Enzyme activity was expressed as µ moles p-nitrophenol released/30 minutes/mg protein.

Lipid peroxidation (LPO): LPO level in testis of control and treated mice were determined by the method of Ohkawa et al., (1979) ³⁰. This method is based on the formation of red chromophore that absorbs at 532nm following the reaction of thiobarbituric acid (TBA) with malonyldialdehyde (MDA) and other breakdown products of peroxidised lipids collectively called as thiobarbituric acid reactive substances (TBARS).

Energy Metabolism Estimation:

Succinate Dehydrogenase Activity (SDH): SDH activity was measured by the method of Beatty et al., (1966) ³¹. The electrons released by the enzyme SDH from the substrate are taken up by an electron acceptor INT which is reduced to red coloured formazan. After extracting it in ethyl acetate the colour intensity was measured at 420 nm against blank. SDH activity was expressed as µg formazan formed/15 min/mg tissue weight.

Adenosine Triphosphatase (ATPase) Activity: The ATPase activity in testis of control and all treated groups of animals was assayed by the method of Quinn and White (1968) ³² while inorganic phosphate liberated was estimated using the method of Fiske and Subbarow (1925) ³³. Readings were taken at 660 nm on a Systronics Digital Spectrophotometer 167.

Measurement of Antioxidant Enzyme Activity:

Superoxide Dismutase Activity (SOD): Activity of SOD in testis of control and treated mice was estimated by modified spectrophotometric method of Kakkar et al., (1984) ³⁴. The formazan formed at the end of reaction indicates presence of enzyme. One unit of enzyme activity is defined as the enzyme concentration required to inhibit 50% of the optical density of chromogen formed in one minute at 560nm under the assay condition.

Catalase activity (CAT): Catalase activity in testis of control and treated mice was assayed by the modified method of Sinha (1972) ³⁵.

Sperm Parameters:

Sperm Count and Sperm Motility: Sperm count and motility in cauda epididymis of control and treated mice was determined using the Neubauer chamber of hemocytometer according to the method of Prasad et al., (1972) ³⁶.

Sperm Viability:

Live: Dead ratio of cauda epididymal sperms was estimated by using the method of Talbot and Chacon (1981) ³⁷.

Sperm Morphology Smears of the sperm suspension were made, allowed to dry in air, fraction and stained with (10:1) 1% Eosin Y (H₂O) and 60 min later slides mounted under a coverslip with permount mounting medium. For each suspension 1000 sperm were examined at 400-fold magnification with blue-green filters; a total of 2000 sperm were thus examined for each group by the method of Wyrobek and Bruce (1975) ³⁸.

Statistics Analysis: All data are presented as mean ± standard error (SE). Statistical analysis was performed using the SPSS software package (version 16.0; SPSS, Inc., Chicago, Illinois, USA). Comparison between groups was made by one way analysis of variance (ANOVA), taking significance at p<0.05, followed by the Student’s t test, taking significance at p<0.01, p<0.005, and p<0.001. Tukey’s honestly significance difference (HSD) post-hoc test was used for comparison among different treatment groups (p<0.05).

RESULTS:

Gravimetric Study (Terminal Body Weight and Tissue Weight):

Body Weight: Mice treated with low dose and moderate dose of CQ for an interval of 14 and 21 days showed no significant reduction in body weight, whereas when high dose was administered, depletion in body weight was witnessed (p<0.005). When curcumin was given as an antidote to the control mice, the results showed negligible variation in body weight, whereas when curcumin was administered along with the higher dose of CQ, reduction observed in the body weight was not significant and the values were comparable to control group in both the durations.

Organ Weight: Low and moderate dose treatment of CQ showed non - significant decrease at 14 days, whereas, high dose (p<0.01) treatment showed significant decrease in the organ weight.

Low dose treatment of CQ showed non - significant decrease at 21 days, whereas moderate (p<0.01) and high dose (p<0.005) treatment showed significant decrease in testis weight. Supplementation of curcumin alone to control animals and along with the high dose of CQ did not show any significant decrease after 14 and 21 days.

Protein Content: Non-significant decrease in the protein content of testis was witnessed in low, moderate and high dose treatment of CQ after 14 days. Low dose and moderate dose of CQ treatment did not show any significant decrease in protein content of testis after 21 days, whereas high dose of CQ resulted in significant decline in protein level of testis (p<0.005). Curcumin when administered to control mice alone and when supplemented along with the high dose of CQ exhibited insignificant decrease in protein content of testis after 14 and 21 days treatment.

Enzymatic Parameters: ACPase activity was found to be non-significant in testis after the low dose and moderate dose treatment, whereas there was significant increase (p<0.01) after the administration of high dose during both the time intervals.

ACPase remained unaltered after administering curcumin alone to control mice during 14 and 21 days. Also, animals treated with CQ and supplemented with curcumin did not show significant change in ACPase activity in testis after 14 and 21 days. ALKpase activity showed no significant change as compared to control after 14 and 21 days treatment of low dose and moderate dose of CQ. However, when high dose of CQ was administered, the ALKpase activity was found to be significantly decreased (p<0.01), (p<0.005) after 14 and 21 days respectively. ALKpase remained unaltered after administering curcumin alone to control mice. Also, animals treated with CQ and supplemented with curcumin did not show significant change in ALKpase activity in testis after 14 and 21 days.

SDH activity did not show any significant change in testis during 14 and 21 days treatment of low dose. Testis SDH activity decreased significantly after the administration of moderate dose (p<0.01) during both the time intervals. High dose of CQ when administered resulted in significant decrease (p<0.01) after 14 days and moderately significant decrease (p<0.005) after 21 days. SDH remained unaltered after administering curcumin alone to control mice. Also, animals treated with CQ and supplemented with curcumin did not show significant change in SDH activity in testis during both the durations.

ATPase activity was found to be non-significant in testis after the low dose and moderate dose treatment during both the time intervals. High dose also when administered was found to be non-significant after 14 days, however, there was significant decrease (p<0.01) after 21 days. ATPase remained unaltered after administering curcumin alone to control mice. Also, animals treated with CQ and supplemented with curcumin did not show significant change in ATPase activity in testis after 14 and 21 days.

Oxidative Parameters: SOD activity was found to be unaltered in testis at low dose treatment of CQ during both the time intervals. When moderate dose of CQ was administered SOD activity was found to be non-significant after 14 days and exhibited significant decline (p<0.005) after 21 days. Also, there was moderately significant (p<0.005) decline at high dose after 14 days and highly significant decline (p<0.001) after 21 days. Curcumin when administered to control mice alone and when supplemented along with the high dose of CQ exhibited insignificant decrease in SOD activity of testis after 14 and 21 days treatment. During both the time intervals, CAT activity was found to be non-significant for each of the three treatments of CQ. Curcumin alone and in combination with the high dose of CQ also showed non – significant decrease in CAT activity of testis after 14 and 21 days treatment.

In both the time interval of 14 and 21 days, LPO levels were found to be non-significant in testis after the low dose. Testis exhibited significant elevation (p<0.005) in LPO levels at moderate dose and highly significant elevation (p<0.001) at high dose of CQ during both the durations. Levels of LPO remained unaltered and changes in values were non - significant when curcumin was administered alone. LPO levels were comparable to control in testis after 14 and 21 days duration of CQ and curcumin co-administration.

Sperm Parameters: Low dose treatment of CQ did not show any significant change in all the sperm parameters like sperm count, sperm motility, sperm viability and sperm morphology at 14 and 21 days.

Moderate dose treatment of CQ showed non-significant changes in all the sperm parameters after 14 days of treatment. However, for 21 days, significant decline only in sperm motility and viability parameters was witnessed, while sperm count and morphology were found to be non-significant. For 14 days, high dose treatment of CQ, sperm count was found to be non-significant, whereas sperm motility and viability was significantly decreased (p<0.01) and sperm morphology was significantly increased (p<0.01). High dose treatment for 21 days of CQ revealed significant decline (p<0.01) in sperm count and moderately significant decline in sperm motility and viability (p<0.005), whereas sperm morphology was found to be significantly increased (p<0.01). Curcumin given to control animals alone and in combination with the high dose of CQ showed non - significant changes in all the sperm parameters after 14 and 21 days treatment.

TABLE 2: BODY WEIGHT AND ORGAN WEIGHT (TESTIS) OF CONTROL AND TREATED ANIMALS AFTER 14 AND 21 DAYS TREATMENT

Values are mean ± S.E.,*p<0.01, **p<0.005, NS = Not significant

 TABLE 3: PROTEIN CONTENT IN TESTES OF CONTROL AND TREATED ANIMALS AFTER 14 AND 21 DAYS TREATMENT

Values are mean ± S.E., **p<0.005, NS = Not significant, Protein – mg/100mg tissue weight

 TABLE 4: ACTIVITIES OF ACPase, ALKPase, SDH AND ATPase IN TESTES OF CONTROL AND TREATED ANIMALS AFTER 14 AND 21 DAYS TREATMENT

Values are mean ± S.E.,*p<0.01, **p<0.005, NS = Not significant

ACPase – μmoles of p-nitrophenol released/mg protein

ALKPase – μmoles of p-nitrophenol released/mg protein

SDH – μg formazan released/15 min/mg protein

ATPase – μmoles i.p. released/30 min/mg protein

TABLE 5: ACTIVITIES OF SOD AND CAT IN TESTES OF CONTROL AND TREATED ANIMALS AFTER 14 AND 21 DAYS TREATMENT

Values are mean ± S.E., **p<0.005, ***p<0.001, NS = Not significant

SOD – units/mg protein

Catalase (CAT) – μmoles H₂O₂ consumed/mg protein

TABLE 6: LEVEL OF LPO IN TESTES OF CONTROL AND TREATED ANIMALS AFTER 14 AND 21 DAYS TREATMENT

Values are mean ± S.E.,*p<0.01, ***p<0.001, NS = Not significant

LPO - nanomoles of MDA/100 mg tissue weight

TABLE 6: SPERM PARAMETERS OF CONTROL AND TREATED ANIMALS AFTER 14 AND 21 DAYS TREATMENT

Values are mean ± S.E.,*p<0.01, **p<0.005, NS = Not significant

DISCUSSION: Chloroquine is still having a good position in malaria therapy as it is the cheapest antimalarial drug and readily available; particularly in the rural setting ³⁹. The results of the harmful effects of the drug on the reproductive functions are very scarce. There was an increased demand of this drug in African countries in the past, especially those which are closer to line of tropics where there is a diversity of vectors. Thus chloroquine has been supplied through governmental health organizations as well as international bodies like WHO from last decade. As a result the endemic populations of these areas are at greater risk of chloroquine toxicity which may affect the reproductive efficacy of the medicating individuals. Hence, the present study is to evaluate effects of CQ on reproductive tissues of male mammal and to mitigate toxicity using a potent antioxidant curcumin.

Literature abounds on the adverse effects of chloroquine on tissues ^{40 - 44} and several studies have been carried out on its antifertility potentials. There are reports suggesting that CQ can be added to the long list of drugs and chemicals causing damage to chromosomes, which is in turn associated with genetic damage ^{45, 46}.

Decreased protein content found post-treatment could be attributed to altered physiology, or impairment in protein synthesis or loss of appetite. Studies have reported that that protein content was decreased when CQ was administered in higher dose (970 mg/kg b.w.), which also resulted in liver damage ²⁷. Magwere et al. (1997) ⁴⁷ have shown that a therapeutic dose of CQ leads to a decrease in protein turnover in humans. Cells which undergo rapid multiplication and protein synthesis are more prone to damage by CQ, as it readily accumulates in such tissues ⁴⁸. Okanlawon and Dyn (1996) ⁴⁹ had reported that CQ increases trans-epithelial resistance in immature Sertoli cells and exhibits anti-proteases activity.

Moreover, significant reduction in body weight as well as testis weight in CQ-treated animals was noted which is concomitant with decrease in protein synthesis. Similar such results have been reported in rats exposed to CQ ⁵⁰. Weight loss was probably a consequence of loss of appetite, thereby reduced intake of food by animals after drug treatment, an evident observation during treatment period. Lysosomes of Sertoli cells incorporate ACPase, which possess heterogenous function ⁵¹. CQ possesses lysomotropic property which induces its uptake in the lysosomes thereby changing its size, leading to increase in size and number of hepatic lysosomes ⁵⁰. Along with this, there is also an elevation in enzyme activities of non-target cells that have been exposed to CQ. Together, all this results in the elevation of ACPase activity in the testis of CQ treated mice. Fredman et al., (1987) ⁴⁸ reported CQ reduces the activity of the lysosomal enzymes while increasing the liver acid phosphatase.

Adverse effects on phosphatase activities are caused by wide range of chemicals including drugs. One of the important groups of enzymes is ALKpases which are omni-present in all the tissues of body, especially cell membranes. Thaker et al., (1997) ⁵² reported that ALKpases carry out the crucial functioning of transport of metabolite(s) across the membranes and are highly vulnerable to damage caused by xenobiotics, which may alter its regular machinery. Thus, CQ which was administered in a dose dependent manner in the present study disturbed the proper functioning of these enzymes thereby causing its impairment and overall decline.

Succinate dehydrogenase (SDH) oxidises succinate to fumarate as a component of the tricarboxylic acid cycle and ubiquinone to ubiquinol in the mitochondrial electron transport chain ⁵³. Any alteration in the morphology and function of mitochondria would hamper the activity of this enzyme. Mitochondrial dysfunction and hampered oxidative metabolism may therefore, be one of the reasons for change in the activity of SDH after undergoing CQ treatment. ATPases are important metabolic regulatory enzymes associated with ATP metabolism. The activities of SDH and ATPase were decreased in a dose-dependent manner, which resulted in impaired energy metabolism. CQ adversely affects mitochondrial energy transduction in vivo by acting as uncouplers of oxidative phosphorylation and lowers the cytochrome a, a₃, and b content ⁵⁴. Liang et al., (2016) ⁵⁵ also showed mitochondrial damage and altered cytochrome C oxidase activities after CQ treatment in rats, which were in support of our study. Superoxide dismutases (SOD) are ubiquitous enzymes in aerobic organisms that function to catalytically convert O₂^•- to oxygen (O₂) and hydrogen peroxide (H₂O₂), the latter is then reduced to water (H₂O) and oxygen (O₂) with the help of catalase ⁵⁶. After the CQ treatment the SOD activity in the testis of mice decreased significantly, whereas LPO increased significantly having a probable role in oxidative stress. SOD thus, acts as a primary line of enzymatic defense and prevents further generation of free radicals ⁵⁷. Magwere et al., (1997) ⁴⁷ demonstrated that the decreased SOD activity in the testis may be due to the accumulation of superoxide radical which consequently increases LPO levels after CQ treatment. Several other studies have also reported that CQ administration leads to increase in LPO levels and lowered enzymatic and non-enzymatic antioxidants ^{47, 58}. The inhibitory effect of CQ on sperm metabolism and regulation of cell survival and motility (measured by production of lactic acid and CO₂) have been also reported ^{15, 59, 60}.

CQ caused disruption of spermatogenesis due to insufficient production of androgens by Leydig cells ⁴³. Investigation by Ashiru et al., (1991) ⁶¹ had shown a reduction in tubular length and diameter of seminiferous tubules and also reported that the injection of rats with CQ for 16 weeks eliminated all Leydig cells. Asuquo et al., (2009) ⁶² also reported degenerative changes in the seminiferous tubules of chloroquine phosphate-treated rats. The elimination of Leydig cells by chloroquine may eliminate testosterone and other Leydig factors that may be required for spermatogenesis ⁶³. Okanlawon and Ashiru (1998) ⁶⁴ showed that in CQ treated rats, there was disruption of spermatogenesis, which was accompanied by a decline in serum concentration of testosterone in the rats. There are reports suggesting CQ and hydroxychloroquine (HCQ) cross the placenta with no significant difference in the mean concentration in maternal and cord blood ⁶⁵. Also, chloroquine is almost completely absorbed into the bloodstream and is concentrated in the tissues, with concentrations being 200 to 700 fold those in plasma ⁶⁶.

Thus, it is possible that spermatozoa in individuals taking long-term chloroquine may be subjected to tissue CQ concentrations approaching those that are capable of producing adverse effects on sperm function.

As there was generation of toxicity due to CQ observed in present investigation, a need to look for an ameliorative agent emerged. Curcumin was opted for as it is considered as the “Indian solid gold” or “spice for life” ¹⁹, and has captured the spotlight in the phytochemical research arena. Due to its incorporation in daily cooking, curcumin becomes an especially important antioxidant due to this ease of access and availability, allowing for maintenance of relatively constant levels in the body and numerous health benefits.

Hence, curcumin was administered along with CQ administration in the present study. Duvoix et al., (2005) ⁶⁷ reported that curcumin, a phenolic compound acts as a chemo-protective agent and protects the cells against the damage caused due to oxidative stress.

Curcumin, when administrated alone and in supplementation with the high dose of CQ, did not show any alterations in the parameters studied, with the values being comparable to the control. However, that does not hinder its property of being a potential candidate for reviving the damage caused by the toxicant. Apart from scavenging the free radicals, curcumin protects against a wide array of degenerative diseases by modulating the biochemical marker enzymes, LPO, and augmenting the antioxidant defense system ^{68, 69}.

Further, Halliwell and Gutteridge (2002) ⁷⁰ highlighted the beneficial effects of curcumin, which included capacity to quickly and efficiently scavenge lipid peroxyl radicals before they attack membrane lipids, thereby lowering oxidative damage.

The protective effect of curcumin on the testis may be explained by the fact that it prevents cellular damage occurring as a result of oxidative stress in spermatogenic cells of seminiferous tubules and Leydig cells of the stroma ⁷¹. Similarly Mohanty et al., (2006) ⁷² found that the role of turmeric in testicular protection may be referred also to its anti-oxidant property. It was also found that curcumin supplementation had prevented chromium-induced decrease in weight of accessory sex organs due to normal serum testosterone level ⁷³. Moreover, curcumin administration to male Wistar rats was able to ameliorate lindane-induced reproductive toxicity in pretreatment, post treatment and combination groups ⁷⁴. Additionally, it was demonstrated that curcumin exerts its protective effect by modulating lipid peroxidation and augmenting antioxidant defense system ⁶⁹. Curcumin may stop peroxidative alteration in the sperm and the testicular membrane which leads to enhancement of sperm motility and decrease in spermatozoa defects ⁷⁵.

Mathuria and Verma (2008) ⁷⁶ investigated the influences of curcumin on aflatoxin-induced toxicity in mice spermatozoa. Aflatoxin noticeably reduced sperm count, viability, and motility; different morphologic defects were encountered; thus, treatment with curcumin improved aflatoxin-induced sperm decrease, immobilization, and viability, and enhanced the morphological characteristics of the sperm. Another study revealed that curcumin protects Leydig cells of mice from the damage caused by chronic alcohol consumption ⁷⁷. Moreover, hepatoprotective effect of tetrahydrocurcumin has been also demonstrated in chloroquine-induced toxicity in rats ²⁷.

Thus, curcumin, due to its ability to affect a wide range of molecular targets and an excellent safety profile, was shown to be a potential candidate for the prevention and/or treatment of a number of diseases. Several reports have demonstrated the protective role of curcumin against many known toxicants which is in support of the ameliorative effects observed in the present study.

CONCLUSION: Based on the present investigation and available review of literature it can be clearly indicated that short term use of Chloroquine to treat malaria could lead to anti-spermatogenic and possible anti-fertility effects, which were noted in the present work as early as 14 and 21 days. Further, curcumin being a natural compound, isolated from the rhizome of turmeric plant, has all the properties of being a wonder drug, and causes no adverse effect even when used in high dose. Present data reveals that administration of curcumin along with chloroquine could prevent the toxic influences of chloroquine and thus may prove to be an effective mitigating agent while treating malaria. As the malaria prone countries are in the developing phase, it will be greatly beneficial if they rely on this natural medicine, as it is cheap and cost-effective.

ACKNOWLEDGEMENT: The authors gratefully acknowledge the laboratory facilities provided by Department of Zoology, Gujarat University (Ahmedabad) and and staff of Statistics for their help in the statistical analysis of the data.

CONFLICT OF INTEREST: The authors declare no conflict of interest related to employment, consultancies, stock ownerships, grants or other funding.

REFERENCES:

1. Breman JG, Alilio MS and Mills A: Conquering the intolerable burden of malaria: what’s new, what’s needed: a summary. American Journal of Tropical Medicine and Hygiene 2004; 71:1-15.
2. James LA and Webb JR: The long shadow of malaria interventions in tropical Africa. The Lancet 2009; 374:1883-1884.
3. Wernsdorfer WH and McGregor I: Malaria: In Principles and Practice of malariology 1988. Churchhill Livingstone Edinburgh; 1128-1179.
4. Wongsrichanalai C, Pickard AL, Wernsdorfer WH and Meshnick SR: Epidemiology of drug-resistant malaria. Lancet Infect. Dis. 2002; 2: 209-218.
5. Sharma V: Therapeutic Drugs for Targeting Chloroquine Resistance in Malaria. Mini Rev. Med. Chem 2005; 5(4): 337-351.
6. Sachs J and Malaney P: The economic and social burden of malaria. Nature 2002; 415: 680-5.
7. Agboruche RL: In-vitro toxicity assessment of antimalarial drug toxicity on cultured embryonic rat neurons, macrophage (RAW 264.7), and kidney cells (VERO-CCl-81). Faseb J 2009; 23:529.
8. Izunya AM, Nwaopara AO and Oaikhena GA: Effect of chronic oral administration of chloroquine on the weight of the heart in Wistar rats. AJMS 2010; 2(3): 127-31.
9. Nduka EU and Dada OA: Inhibition of gonadotropin and prostaglandin stimulation of testicular steroidogenesis in malnourished rats. Andrology 1984; 16:406-409.
10. Solomon VR and Lee H: Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol 2009; 625: 220-33.
11. Harrison ST and Laufer MK: Antimalarial drug resistance in Africa: key lessons for the future. Ann N Y Acad Sci. 2015; 1342: 62–67.
12. Syu JS, Baba T, Huang JY, Ogawa H, Hsieh CH, Hu JX, Chen TY, Lin TC, Tsuchiya M, Morohashi KI and Huang BM: Lysosomal activity maintains glycolysis and cyclin E1 expression by mediating Ad4BP/SF-1 stability for proper steroidogenic cell growth. Scientific reports. 2017; 21; 7(1): 240.
13. Browning DJ: Hydroxychloroquine and chloroquine retinopathy. Springer; 2014; 9.
14. El Shishtawy MA, Hassan KH, Ramzy R, Berri F, Mortada M, Nasreddine S and Ezzedine M: Comparative toxicity study of chloroquine and hydroxychloroquine on adult albino rats. European Scientific Journal, ESJ. 2015; 24; 11(3):
15. Samplaski MK and Nangia AK: Adverse effects of common medications on male fertility. Nature Reviews Urology. 2015; 12(7): 401.
16. Desai KR, Dattani JJ, Rajput DK, Moid N, Highland HN and George LB: Role of curcumin on chloroquine phosphate-induced reproductive toxicity.Drug and chemical toxicology 2012; 35(2):184-191.
17. Greenwell M, Rahman PK: Medicinal plants: their use in anticancer treatment. International journal of pharmaceutical sciences and research. 2015; 6(10): 4103.
18. Singh R, Kumar M, Mittal A and Mehta PK: Microbial metabolites in nutrition, healthcare and agriculture. 3Biotech. 2017; 7(1): 15.
19. Aggarwal BB, Sundaram C, Malani N and Ichikawa H: Curcumin: the Indian solid gold. Adv Exp Med Biol 2007; 595: 1-75.
20. Chattopadhyay I, Biswas K, Bandyopadhayay U and Banerjee RK: Turmeric and curcumin: biological actions and medicinal applications. Curr. Sci. 2004; 87:44-53.
21. Araujo CC and Leon LL: Biological activities of Curcuma longa Mem. Inst. Oswaldo Cruz 2001; 96:723-738.
22. Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK and Lee SS: Molecular mechanisms underlying chemoprotective activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-ҡB activation. Mutat. Res. 2001; 243-268.
23. Chainani-Wu N: Safety and anti-inflammatory activity of curcumin: a component of turmeric (Curcuma longa). J. Altern. Complement Med. 2003; 9:161-168.
24. Okada K, Wangpoengtrakul C, Tanaka T, Toyokuni S, Uchida K and Osawa T: Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice. J. Nutr. 2001; 131: 2090-2095.
25. Rukkumani R, Aruna K, Varma PS, Rajesekaran KN and Menon VP: Comparative effects of curcumin and an analog of curcumin on alcohol and PUFA induced oxidative stress. J. Pharm. Pharm. Sci. 2004; 7: 274-283.
26. Walum E: Acute Oral Toxicity Environmental Health Perspectives 1998; 106:497-503.
27. Pari L and Amali DR: Protective role of tetrahydro curcumin (THC) an active principle of turmeric on chloroquine induced hepatotoxicity in rats. J Pharm Sci 2005; 8:115-123.
28. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein measurement with folin phenol reagent. J Biol Chem 1951; 193:265–275.
29. Bessey OA, Lowry OH and Brick NJ: A method for the rapid determination of acid and alkaline phosphatases in 5 cu mm of serum. J Biol Chem 1946; 164:321-325.
30. Ohkawa H, Ohishi N and Yagi K: Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Anal Biochem 1979; 95:351–358.
31. Beatty CH, Basinger GM, Dully CC and Bocek RM: Comparision of red and white voluntary skeletal muscle of several species of primates. J Histochem Cytochem 1966; 14: 590–600.
32. Quinn PJ and White IG: Distribution of adenosine triphosphatase activity in Ram and bull spermatozoa. J Reprod Fertil 1968; 15: 449-452.
33. Fiske CH and Subbarow Y: The colorimetric determination of phosphorus. JBC 1925; 66(2): 375-400.
34. Kakkar P, Das B and Viswanathan P N: A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984; 4:130–132.
35. Sinha AK: Colorietric assay of catalase. Anal. Biochem. 1972; 47:389-394.
36. Prasad MRN, Chinoy NJ and Kadam KM: Changes in succinic dehydrogenase levels in rat epididymis under normal and altered physiological condition. Fertil Steril 1972; 23:186-190.
37. Talbot P and Chacon P: A triple stain technique for evaluating normal acrosome reaction of human sperm. J Exp Zool 1981; 215: 210-208.
38. Wyrobek AJ and Bruce WR: Chemical induction of sperm abnormalities in mice (25 chemicals/ mutagens/ teratogens / carcinogens). Proc. Nat. Acad. Sci. USA. Cell Biol. 1975; 72(11): 4425-4429.
39. Jimmy EO, Achelonu E and Orji S: Antimalaria dispensing pattern by patent medicine dealers in rural settlement in Nigeria. Journal of Public Health, England 2000; 114: 282-285.
40. Martins AC, Cayotopa AD, Klein WW, Schlosser AR, Silva AF, Souza MN, Andrade BW, Filgueira-Júnior JA, Pinto WD and da Silva-Nunes M: Side effects of chloroquine and primaquine and symptom reduction in malaria endemic area (Mâncio Lima, Acre, Brazil). Interdisciplinary perspectives on infectious diseases. 2015.
41. Al-Bari MA: Chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases. JA C 2015; 70(6): 1608-21.
42. Giovanella F, Ferreira GK, PRÁ SD, Carvalho-Silva M, Gomes LM, Scaini G, Gonçalves RC, Michels M, Galant LS, Longaretti LM and Dajori AL: Effects of primaquine and chloroquine on oxidative stress parameters in rats. Anais da Academia Brasileira de Ciências. 2015; 87(2): 1487-96.
43. Rodriguez-Caruncho C and Marsol IB: Antimalarials in dermatology: mechanism of action, indications, and side effects. Actas Dermo-Sifiliográficas (English Edition). 2014; 105(3): 243-52.
44. Chaanine AH, Gordon RE, Nonnenmacher M, Kohlbrenner E, Benard L, Hajjar RJ: High‐dose chloroquine is metabolically cardiotoxic by inducing lysosomes and mitochondria dysfunction in a rat model of pressure overload hypertrophy. Physiological reports. 2015; 3(7):
45. Hu T, Li P, Luo Z, Chen X, Zhang J, Wang C, Chen P and Dong Z: Chloroquine inhibits hepatocellular carcinoma cell growth in vitro and in vivo. Oncology reports. 2016; 35(1): 43-9.
46. Roy LD, Mazumdar M, Giri S: Effects of low dose radiation and vitamin C treatment on chloroquine‐induced genotoxicity in mice. Environmental and molecular mutagenesis. 2008; 49(6): 488-95.
47. Pari L and Murugan P: Tetrahydrocurcumin: Effect on Chloroquine‐Mediated Oxidative Damage in Rat Kidney. Basic and clinical pharmacology and toxicology. 2006; 99(5): 329-34.
48. Fredman P, Klinghart GW and Svennerholni L: Effect of chloroquine on the activity of some lysosomal enzymes involved in ganglioside degradation. Biochim. Biophys. Acrtr 1987; 917:1-8.
49. Okanlawon A, Kusemiju O and Dym M: Direct toxic effects of clinical doses of chloroquine on transferrin secretion in immature rat sertoli cells in vitro. The Nigerian postgraduate medical journal. 2001; 8(4): 165-9.
50. Zahid A and Abidi TS: Effect of chloroquine on liver weight of developing albino rats. J Pak Med Assoc 2003; 53: 21-23.
51. Chemes H: The phagocytic function of Sertoli cells: a morphological, biochemical, and endocrinological study of lysosomes and acid phosphatase localization in the rat testis. Endocrinology 1986; 119:1673-1681.
52. Thaker J, Chhaya J, Nuzhat S, Mittal R, Mansuri AP and Kundy R: Dose and duration dependent toxicity of Cr on acid-alkali phosphatase in six vital organs of mud skipper periophtalamus dipes. Indian JEB 1997; 35: 397-440.
53. Huang S and Millar H: Succinate dehydrogenase: the complex roles of a simple enzyme. Current Opinion in Plant Biology 2013; 16(3): 344-349.
54. Katewa SD and Katyare SS: Treatment with antimalarials adversely affects the oxidative energy metabolism in rat liver mitochondria. Drug Chem Toxicol 2004; 27:41–53.
55. Liang DH, Choi DS, Ensor JE, Kaipparettu BA, Bass BL and Chang JC: The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair. Cancer letters. 2016; 376(2): 249-58.
56. Samuel EL, Marcano DC, Berka V, Bitner BR, Wu G, Potter A, Fabian RH, Pautler RG, Kent TA, Tsai AL and Tour JM: Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters. Proceedings of the National Academy of Sciences. 2015; 112(8): 2343-8.
57. El-Demerdash FM, Yousef MI and Radwan FM: Ameliorating effect of curcumin on sodium arsenite-induced oxidative damage and lipid peroxidation in different rat organs. Food Chem Toxi 2009; 47: 249-54.
58. Murugavel P and Pari L: Attenuation of chloroquine induced renal damage by α-lipoic acid: possible antioxidant mechanism. Ren Fail 2004; 26: 515-522.
59. Aparicio IM, Espino J, Bejarano I, Gallardo-Soler A, Campo ML, Salido GM, Pariente JA, Pena FJ and Tapia JA: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility. Scientific reports. 2016; 6: 33647.
60. Okanlawon AO, Noronha CC and Ashiru OA: An investigation into the effects of chloroquine on fertility of male rats. W. African J. Med. 1993; 12:118-121.
61. Zhang J, Zhang X, Liu Y, Su Z, Dawar FU, Dan H, He Y, Gui JF and Mei J: Leucine mediates autophagosome-lysosome fusion and improves sperm motility by activating the PI3K/Akt pathway. Oncotarget. 2017; 8(67): 111807.
62. Asuquo OR, Igiri AO, Akpaso MI and Akpanabiatu MI: Histopathological and Biochemical effects of chloroquine phosphate on the testis of male albino Wister rats. Internet Journal of Laboratory Medicine 2009; 4(2):
63. Salman TM, Olayaki LA, Shittu ST and Bamgboye SO: Serum testosterone concentration in chloroquine treated rats: effects of ascorbic acid and alphatocopherol. African Journal of Biotechnology. 2010; 9(27): 4278-81.
64. Sahu R, Kashyap P: Genotoxic potential of some commonly used antimalarials: A review. International Journal of Pharmaceutical Sciences and Research. 2012; 3(6): 1569.
65. Costedoat-Chalumeau N, Amoura Z, Aymard G, Huong DLT, Wechsler B, Vauthier D, Dermer ME, Darbois Y and Piette JC: Evidence of transplacental passage of hydroxychloroquine in humans. Arthritis Rheum 2002; 46: 1123-1124.
66. Gilman AG, Goodman LS, Rall TW and Murad F: The Pharmacological Basis of Therapeutics. Macmillan Publishing Co., London, Seventh Edition 1985.
67. Duvoix A, Blasius R, Delhalle S, Schnekenburger M, Morceau F and Henry E, Dicato M and Diederich M: Chemopreventive and therapeutic effects of curcumin. Cancer Lett 2005; 223: 181-190.
68. Kalpana C and Menon VP: Modulatory effects of curcumin on lipid peroxidation and antioxidant status during nicotine-induced toxicity. Pol. J. Pharmacol. 2004; 56: 581-586.
69. Manikandan P, Sumitra M, Aishwarya S, Manohar BM, Lokanadam B and Puvanakrishnan R: Curcumin modulates free radical quenching in myocardial ischaemia in rats. Int J Biochem Cell Biol 2004; 36: 1967-1980.
70. Halliwell B and Gutteridge JMC: Free radicals in biology and medicine. New York: Oxford University Press, Vol. III, 2002: 105-245.
71. Aly HAA, Mansour AM, Abo-Salem OM, Abd-Ellah HF and Abdel-Naim AB: Potential testicular toxicity of sodium nitrate in adult rats. Food and Chemical Toxicology 2009; 48:572-578.
72. Mohanty I, Arya S and Gupta SK: Effect of Curcuma longa and Ocimum sanctum on myocardial apoptosis in experimentally-induced myocardial ischemic–reperfusion injury. BMC Complement. Altern. Med. 2006; 6: 3-14.
73. Chandra AK, Chatterjee A, Ghosha R and Sarkar M: Effect of curcumin on chromium-induced oxidative damage in male reproductive system. Environmental Toxicology and Pharmacology 2007; 24:160-166.
74. Sharma P and Singh R: Protective Role of Curcumin on Lindane Induced Reproductive Toxicity in Male Wistar Rats. Bull Enviro Contam Toxicol. 2010; 84: 378-384.
75. Farombi EO, Abarikwu SO, Adedara IA and Oyeyemi MO: Curcumin and Kolaviron Ameliorate Di-n-Butylphthalate-Induced Testicular Damage in Rats. Basic & Clinical Pharmacology Toxicol 2007; 100(1):43-8.
76. Mathuria N and Verma RJ: Curcumin ameliorates aflatoxin-induced toxicity in mice spermatozoa. Fertil Steril 2008; 90(3):775-80.
77. Giannessi F, Giambelluca MA, Grasso L, Scavuzzo MC and Ruffoli R: Curcumin protects Leydig cells of mice from damage induced by chronic alcohol administration. Med Sci Monit 2008; 14(11): 237-42.
    

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    Desai KR, Patel PB and Siddiqui Z and Highland HN: Sub-acute chloroquine toxicity on testis of Swiss albino mice and its amelioration by Curcumin. Int J Pharm Sci Res 2018; 9(8): 3235-46. doi: 10.13040/IJPSR.0975-8232.9(8).3235-46.

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