ASSESSMENT OF PROTECTIVE EFFECT OF CURCUMIN ON CADMIUM CHLORIDE INDUCED TESTICULAR TOXICITY IN MALE SWISS ALBINO MICE

P. Singh. It is a well established fact that cadmium is a known clastogen, mutagen and a carcinogen. Its ingestion results in long term multifaceted alterations and impaired biological and physiological functions. It is also known that curcumin a mandatory dietary condiment has potent healing and curative properties. Hence the aim of the present study was to investigate the protective effects of curcumin against cadmium chloride induced testicular toxicity in male Swiss albino mice (Mus musculus). Male Swiss albino mice were divided into four groups, each comprising of six mice. (a) Control group administered only vehicle; (b) experimental group administered only curcumin (10mg/animal/day); (c) experimental group administered a single oral dose of cadmium chloride (50mg/kg/animal/day) and (d) experimental group administered curcumin (10mg/animal/day) for 15 days and on the 16th day cadmium chloride (50mg/kg/animal/day). After completion of dose administration mice were sacrificed and testes were excised. Sections of testes were processed for histopathological, histochemical and biochemical studies. Histopathological examination showed cadmium induced toxicity as manifested by damaged disorganized cellular layers of seminiferous tubules, leydig cells, apoptotic cells, and loss of interstitial tissue ameliorated by the administration of Curcumin. Histochemcial staining resulted in altered distribution pattern of lactate dehydrogenase (LDH) enzyme on cadmium administration. The curcumin pre treated group showed an enzymatic distribution profile similar to that of control group. Biochemical test result in a marked decrease in the activity levels of the lactate dehydrogenase (LDH) significantly (P<0.05) in the cadmium treated mice. The degree of decrease of these enzymes was significantly less (P<0.05) when mice were pre treated with curcumin. Curcumin appear to possess an ameliorative potential against cadmium-induced testicular toxicity.


P. Singh.
It is a well established fact that cadmium is a known clastogen, mutagen and a carcinogen. Its ingestion results in long term multifaceted alterations and impaired biological and physiological functions. It is also known that curcumin a mandatory dietary condiment has potent healing and curative properties. Hence the aim of the present study was to investigate the protective effects of curcumin against cadmium chloride induced testicular toxicity in male Swiss albino mice (Mus musculus). Male Swiss albino mice were divided into four groups, each comprising of six mice. (a) Control group administered only vehicle; (b) experimental group administered only curcumin (10mg/animal/day); (c) experimental group administered a single oral dose of cadmium chloride (50mg/kg/animal/day) and (d) experimental group administered curcumin (10mg/animal/day) for 15 days and on the 16th day cadmium chloride (50mg/kg/animal/day). After completion of dose administration mice were sacrificed and testes were excised. Sections of testes were processed for histopathological, histochemical and biochemical studies. Histopathological examination showed cadmium induced toxicity as manifested by damaged disorganized cellular layers of seminiferous tubules, leydig cells, apoptotic cells, and loss of interstitial tissue ameliorated by the administration of Curcumin. Histochemcial staining resulted in altered distribution pattern of lactate dehydrogenase (LDH) enzyme on cadmium administration. The curcumin pre treated group showed an enzymatic distribution profile similar to that of control group. Biochemical test result in a marked decrease in the activity levels of the lactate dehydrogenase (LDH) significantly (P<0.05) in the cadmium treated mice. The degree of decrease of these enzymes was significantly less (P<0.05) when mice were pre treated with curcumin. Curcumin appear to possess an ameliorative potential against cadmium-induced testicular toxicity.

Results:-
Histopathological profile of testes:-Histoarchitecture of control and Curcumin treated groups displayed normal morphological profile round seminiferous tubules, compact interstitial connective tissue and germ cells (FIG. 1). In cadmium treated mice (50mg/kg) there was no significant alteration in the oval shape of testis. However, in certain areas induction of hemorrhage in the testicular vasculature was observed. The significant alterations in testicular histoarchitecture, where there was a distinctive variation in the shape of seminiferous tubules which changed from typical round to irregular and testis of Cadmium treated groups showing damaged seminiferous tubules and Leydig cells. Loss of interstitial connective tissue was observed (FIG.1 and FIG.2). In cadmium treated animal due to loss of germinal cells there was a significant expansion in the width of seminiferous tubular lumen which appeared to be time dependent (FIG.3).
As compared to only cadmium treated group a decline in the area of hemorrhage in the testicular vasculature was observed in pre treated group. The loss of cell contact and initiation of exfoliation appeared to be checked and the comparative damage was significantly less. There was no total disruption of cellular association were observed (FIG.4).   TABLE I. Strong staining is evident in control and Curcumin treated groups, spermatogonia mother cells displayed light to moderate enzyme reactions. As these grow and enter premeiotic prophase, the LDH staining was elevated. The spermatogonia and spermatids in various phases of differentiation exhibited intense enzyme activity The sertoli cells also showed moderate LDH activity. Except for leydig cell which showed intense LDH reaction, the other interstitial cells and tunica did not show any reaction (FIG.5 (a)).
Cadmium chloride (50 mg/kg/ animal for a day) treated group showed differential LDH reaction in various cells of the testes as compared to control. Spermatogonia mother cells and spermatogonia did not show any reaction whereas moderate reaction was seen in spermatids. Light reaction was evinced in sertoli cells and leydig cells whereas in other cells of interstitium and tunica negligible reaction was showed (FIG.5 (b)).
Cur cumin pre treated group displayed light reactions in spermatogonia mother cells. The LDH staining was elevated in spermatogonia. The spermatids in various phases of differentiation exhibited moderate enzyme activity. The sertoli cells and Leydig cells also showed light LDH activity, the other interstitial cells and tunica showed moderate reaction (FIG.5 (c)).   Table II. The estimates of LDH activity in testes calculate according to the following formula: LDH(U/L) = (ΔAbs/min) x factor, ε NAD/NADH = 6230 M -1 cm -1 LDH activity in cadmium treated group was significantly lower than the activity in the control group testes (p < 0.05). However a significant increased in LDH activity in pre treated with curcumin groups when compared with control and cadmium treated groups (FIG.6, table II). Cur cumin + CdCl 2 3379.587 ± 1.115 *,** Values are Means ± SEM of 6 animals for each group. Values bearing superscript are significantly different by ANOVA at p≤ 0.05. *: when compared different groups with control group.; **: when compared (cur+CdCl 2 ) with cadmium chloride group.

Discussion:-
The process of spermatogenesis is a highly sensitive phenomenon depending upon an interplay of a number of enzymes and hormones. Any disturbance in the micro and macro environment of the testis results in the perturbation in sperm production resulting in infertility problems. The management of infertility problems has become an increasingly important part of health services during the past decades. The importance of drugs from plant origin, as fertility regulating agents for the males has long been recognized. Medicinal plants present a repertoire capable of providing varied constituents which could be helpful in infertility management. Curcumin, a potent antioxidant compound found in turmeric, has been used for centuries as a natural dye, seasoning and medicine. In Ayurveda, a 5000 year old system of medicine originating in India, curcumin in turmeric has been used to treat a number of common conditions. Hence, in the present experiment an effort has been made to observe ameliorative effects of curcumin on testicular damage induced by cadmium chloride.
Moreover, cadmium being a metal has been recognized as a reproductive toxicant and has been reported to reduce male fertility and altered sexual behaviour in both humans and rodents (Thomas and Brogan, 1983 (Prozialeck et al., 2006). Acute doses of cadmium are known to have a destructive action on testicular tissue (Parizek, 1960;Gunn and Gould, 1970;Friberg et al., 1974). Necrosis, hemorrhage in testicular vasculature and testicular oedema as observed in the present study which has been also reported in number of earlier studies (Madlafousek, 1971 They observed the activities of lactate dehydrogenase isozyme X (LDH X) decreased in the middle and high dose groups singificantly. These suggest cadmium toxicity on male reproductive system is associated to activities of testicular enzymes. Jianguo et al. (1996) observed LDH-X activity of seminiferous tubules of mouse testis by histochemical method after cadmium and cadmium plus zinc administration. They showed that LDH-X activity had no difference between the control group and the group of cadmium administration for 1 hour, but the activity reduced significantly after cadmium administration for 3 hours (P<0.05),and highly significantly reduced for 6,12 hours(P<0.01)。The results suggest that LDH-X activity of seminiferous tubules of mouse testes reduces in acute cadmium poisoning. Donatus et al. (1990) reported the cytoprotective effect of curcumin in rat hepatocytes. At low concentrations curcumin was found to protect significantly against paracetamol-induced lipid peroxidation, without protection against paracetamol-induced LDH-leakage. At a 100 times higher concentration of curcumin the observed protective effect on LDH-leakage. Padmaja and Raju (2005) reported that selenium administration resulted in a marked decrease in the activity levels of the liver lactate dehydrogenase in the Wistar rat. The degree of decrease of these enzymes was significantly less (P<0.001) when rats were treated with curcumin, a natural constituent of Curcuma longa. Curcumin seems to prevent oxidative damage mediated through selenium and protect the dehydrogenases possibly through its antioxidative property. Sadik (2008) reported that harmful effect of cadmium on testis manifested by germ cell degeneration and impairment of testicular enzymes. Male adult Wistar rats treated with cadmium (2.5 mg/kg body wt, five times a week for 4 weeks) showed decreased testicular enzymes, such as lactate dehydrogenase (LDH) after cadmium exposure. Zoheb et al. (2014) assessed the toxicity of mixture of six metals including lead (Pb), arsenic (As), cadmium (Cd), mercury (Hg), iron (Fe), and copper (Cu) at environmentally realistic concentrations and to determine whether curcumin was able to prevent testicular injury in rats. Administered curcumin (100 mg/kg body wt.), 10 x mixtures, 100 x mixtures, 10x plus curcumin, and 100x plus curcumin. Testes were collected for the analysis of oxidative stress markers, testicular enzymes, and histopathology.Exposure of 10x and 100x mixture significant decrease in LDH activity at 100x exposure level were observed. Lactate dehydrogenase activity was comparable amongst all groups.
El-Fattah et al. (2015) reported the protective role of resveratrol and curcumin on oxidative testicular damage induced by di-(2-ethylhexyl) phthalate (DEHP). Male Wistar rats were given either resveratrol (80 mg/kg BW) or curcumin (200 mg/kg BW) orally for 30 days before and 45 days after DEHP administration. Oxidative damage was observed lactate dehydrogenase (LDH) showed severe declines. Histopathological observations provided evidence for the biochemical and molecular analysis. These DEHP-induced pathological alterations were attenuated by pretreatment with resveratrol and curcumin. Lonare et al. (2015) reported the toxic effects of imidacloprid (IM) on male reproductive system and ameliorative effect of curcumin (CMN) in male Wistar rats. IM (45 and 90 mg/kg, body weight) and CMN (100 mg/kg, body weight) were administered orally to the rats either alone or in combinations for a period of 28 days. IM treatments resulted in significant decrease (p<0.05) lactate dehydrogenase-x. However, the reproductive toxicity parameters, oxidative stress indicators, and histopathological changes were minimized and functional restorations were noticed by co-administration of CMN in IM-treated rats. Thus, on the basis of above findings it can be concluded that cadmium chloride induce perturbations in the normal histology of testis that are ameliorated on pre treatment with curcumin as curcumin thwart per oxidative changes in the testis. Moreover, it can also be suggested that everyone who is supplementation of curcumin led to the improvement in altered structure of testis due to cadmium chloride intoxication. This mitigating effect can be attributed to the potential of curcumin in inducing antioxidant defense mechanisms reported in earlier studies (El-Bahr and Salama 2007; El- Wakf et al., 2011). These observations are similar to the research findings of several researchers who have reported that curcumin has the innate potential to counterbalance reproductive damage induced by heavy metals viz. chromium ( Chandra et al., 2007) and arsenic (Demerdash et al., 2009) .

Conclusion:-
In present study, concluded that where testes is greatly targeted to damage by cadmium intoxication. Along with evidence derived from present study, where exposure to cadmium constitutes a great threat being associated with reproductive injurious effects. Hence, concern should be directed to limit the inadvertent incorporation of cadmium in human body. The curcumin has the ameliorative potential to prevent cadmium induced testicular toxicity.