GLUTATHIONE AND ANTIOXIDANT STUDY IN TESTES OF WISTAR RATS AFTER DIETARY COPPER DEFICIENCY

GLUTATHIONE AND ANTIOXIDANT STUDY IN TESTES OF WISTAR RATS AFTER DIETARY COPPER DEFICIENCY

Aastha Saini, Ankita Rajendra Kurup and Neena Nair *

Department of Zoology, Centre for Advanced Studies, University of Rajasthan, Jaipur, India.

ABSTRACT: Copper is an essential trace element involved in a plethora of functions being a component of several enzymes. Copper deficiency affects people of all age groups, including infants, children, pregnant women, and men. Deficiency occurs due to numerous factors hindering the absorption/uptake of copper. Male Wistar rats (35-50 gm) were divided into four groups: Group 1: Negative control – fed with standard feed (Ashirwad Industries, Chandigarh) and tap water was provided ad libitum .Group 2: Copper control group- Fed with diet containing 126 nmol Cu/gm. Tap water was provided ad libitum. (c) Group 3: Pair-fed group - Fed with 126 nmol Cu /gm diet but the amount of feed given was equal to the feed consumed by the copper-deficient group the previous day to account for stress and starvation effect caused due to low intake diet. Tap water was provided ad libitum (d) Group 4: Copper deficient- animals were fed with 6.3 nmol Cu / gm diet, and demineralized water was provided ad libitum. The experiment was conducted for 2-, 4- and 6 weeks. Glutathione concentration and Cu-Zn SOD decreased significantly (P<0.05) after dietary copper deficiency. After copper deficiency, a significant increase (P<0.05) in total SOD, Mn-SOD, and catalase activities /expression was observed. The vulnerability of testes to oxidative stress and subsequent generation of reactive oxygen species accounts for impaired glutathione concentration and antioxidant enzymes. This indicates that prolonged perturbation of the copper homeostasis would hamper the functional aspect of testes ultimately leading  to infertility.

Keywords: Copper deficiency, Testes, glutathione, Total SOD, Cu-Zn SOD, Mn SOD, Catalase

INTRODUCTION: Micronutrient deficiency is a global problem, especially where the intake of food is cereals which are already low in micronutrients. The problem is further enhanced with these cereals grown in deficient soil, which further reduces its concentration. Copper deficiency is on the rise because soils do not respond to copper added in fertilizers and range to 10- 15% in India ¹. Fodders have been reported to have low copper content, which would impact animals and ultimately human beings ^{2, 3}.

Kohlaa et al.,⁴ reported breastfeeding a full-term female infant with severe copper deficiency. Deficiency of copper can also occur due to antagonistic action of molybdenum, sulfur, iron, excess zinc, malabsorption, foregut surgery, and hereditary disorders ^{5, 6, 7}.

Several authors ^{8, 9} reported average daily intakes of copper in the range of 0.8 - 3 mg, with the absorbed copper being bound to albumin, histidine/glutathione. Copper has the ability to react with superoxide anion and hydrogen peroxide leading to the formation of hydroxyl radical ^{10, 11}. Oxidative stress generation of excess reactive oxygen species (ROS) is one of the major causes of male infertility. Enhanced ROS has profound effects on cells, Gene expression and genetic integrity of spermatozoa ^{12, 13} with an ability to induce lipid peroxidation, damage DNA, RNA, and functions of testicular cells ¹⁴. In testes, Sertoli cells and spermatozoa have been reported to generate reactive oxygen species ^{15, 16}. Oxidative damage in testes has been reported to occur due to prolonged exposure to stress ^{17, 18}. To eliminate these toxic effects mammals / aerobic organisms have efficient mettaloenzymes glutathione peroxidase, superoxide dismutase and catalase ^19-22. Glutathione (GSH), a tripeptide of L- γ –glutamyl –L–cysteinyl glycine abundantly found in mammalian tissues in the range of approximately 0.1-15 mM and has the potential to reduce the level of reactive oxygen species as well as reactive nitrogen species ^23-27.

Most of the copper absorbed by the body is bound mainly to metallothionein glutathione ²⁸. Glutathione - hydrophilic antioxidant functions to (i) neutralize hydrogen peroxide (ii) secondary lipid peroxidation products formed during damage to the tissue ^{29, 30} and (iii) involved in signaling and also scavenges NO ^{31, 32, 33}. Copper, an important component of superoxide dismutase  (SOD, EC1.15.1.1) considered as the first line of antioxidant defense ³⁴, has a  significant role in male reproductive organs protecting the cellular structure and function against oxidative damage. Trist et al.,¹¹ observed that SOD 1 protein provides approximately 90% of total SOD activity in mammalian somatic cells. However, Gu et al.,³⁵ reported   0.73, 0.80 and 0.93 kB SOD1 transcript in mice testes with 0.80 and 0.93 Kb testicular- tSod1 transcripts specific only for the germ cells. Cu/Zn SOD protects against oxidative stress by catalyzing the dismutation of superoxide generated under normal aerobic metabolism. Both transition metals are required for their catalytic activity; although zinc no doubt can be replaced by other metals but copper is critical for the functioning of the enzyme ³⁶.

Intense Cu/Zn SOD protein expression was observed in spermatogonia of human testes, although the expression was weak in other germ cells and somatic cell – Sertoli cells ³⁷. Manganese (Mn), a component of SOD and psuedocatalase has a role in the redox process ³⁸. Catalase (EC 1.11.1.6) catalyzes the dismutation of H₂O₂ – harmful reactive species to oxygen and water, although H₂O₂ also functions as a second messenger involved in altered morphology, proliferation, signaling and apoptosis ³⁹. The metabolization of H₂O₂ occurs both by catalase as well as glutathione peroxidase ^{10, 40}. The study evaluates the effect of dietary copper deficiency on testes of Wistar rats.

MATERIALS AND METHODS:

Synthetic Diet: The diets were formulated by using ICN Research Diet Protocol (1999). The ingredients of the diet (per kg diet) were-  Egg white/ albumin- 180 gm, Corn oil- 100 gm, Corn starch- 443 gm, Sucrose- 200 gm, Cellulose -30 gm, Choline chloride- 2 gm, DL-methionine- 7 gm, AIN- 76 salt mixture- 35 gm, AIN- 76C vitamin-antibiotic mixture- 10 gm. Copper contents of basal diet for each group were estimated at 324.8 nm in air acetylene flame on GBC 902 atomic absorption spectrophotometer, and copper concentrations were adjusted to 126 nmol/gm and 6.3 nmol/gm of copper sulfate.

Experimental Protocol: Male Wistar rats (30-40 days- prepubertal period; 35-50 gm ) were divided into four groups with 10 animals in each group: Group 1: Negative control – fed with standard feed (Ashirwad Industries, Chandigarh) and tap water was provided ad libitum. Group 2: Copper control / CC group- Fed with diet containing 126 nmol Cu/gm. Tap water was provided ad libitum. (c) Group 3: Pair-fed group / PF group- Fed with 126 nmol Cu /gm diet, but the amount of feed given was equal to the feed consumed by the copper-deficient group the previous day to account for stress and starvation effect caused due to low intake diet. Tap water was provided ad libitum (d) Group 4: Copper deficient / CD group- animals were fed with 6.3 nmol Cu / gm diet and demineralized water was provided ad libitum. Male Wistar rats were housed in isolation in polypropylene cages with stainless steel grills.

Cages and water bottles were washed with detergent solution, demineralized water, and rinsed in 1% EDTA solution prepared in demineralized water to remove copper traces. Experiments were conducted for 2-, 4- and 6-weeks and approved by the University Department Ethics Committee and Committee for Control and Supervision of Experiments on Animals [CPCSEA (1678/GO/Re/S/12)]. After completing the experiments, animals were anesthetized; testes were excised, cleaned off of extraneous tissue, weighed on Sartorius BS 124 S electronic balance, and processed for biochemical studies.

Biochemical Studies: Testes were homogenized in Remi tissue homogenizer and centrifuged at 2000 rpm (Remi –R8C laboratory centrifuge) or 10,000 rpm (Sigma refrigerated high-speed centrifuge 4 K15 using 18015 rotar) to remove cell debris. Glutathione (GSH) ^{41, 42}. Superoxide Dismutase (SOD): (A) Total SOD ⁴³, (B) Cu-Zn SOD ⁴⁴ and (C) Mn-SOD (Total SOD –Cu-Zn SOD) and Catalase ⁴⁵ were analyzed. The absorbance was read on Systronics spectrophotometer 169 (Serial No. 827).

Statistical Analysis: Data expressed as mean ± SEM. One way Analysis of Variance (ANOVA) was carried out separately for 2-, 4- and 6- week experimental groups and post- hoc test (Tukey’s Multiple Comparison test) was carried out if the difference was found to be significant. Data were analyzed using Graph Pad Prism Version 7.0e. P < 0.05 was examined to be significant.

RESULT: Glutathione concentration decreased significantly (P<0.05) after 2-, 4- and 6- weeks of the experiment when copper deficient (CD) groups were compared with their respective negative control (NC) , copper control (CC), and pair-fed (PF) groups. The decrease was also significant when pair-fed groups of 2- and 6-week experiments were compared with respective NC and CC groups. However, after 4 weeks the decrease was non-significant compared to NC and CC groups Table 1.

TABLE 1: BIOCHEMICAL ANALYSIS IN TESTES OF WISTAR RAT AFTER 2-,4- AND 6- WEEKS OF  DIETARY COPPER-DEFICIENT  DIET   (MEAN ± SEM)

a= NC Vs CC, b = NC vs PF, * P<0.05 Significantc= NC vs CD, d=CC Vs PF, e =CC Vs CD, f =PF Vs CD Multiple comparison procedures were performed for 2-, 4- and 6 weeks experimental groups.

After 2-, 4- and 6- weeks of dietary copper deficiency total SOD activity increased significantly (P<0.05)  when copper-deficient groups were compared with their respective NC, CC and PF groups. Comparison of three experimental PF groups with respective NC and CC groups revealed a significant increase (P<0.05) Table 1. Cu-Zn SOD activity decreased (P<0.05) significantly after 2-, 4- and 6- weeks of dietary copper deficiency when copper-deficient groups were compared with their respective NC.CC and PF groups. The decrease was also significant (P<0.05) when PF groups( 2-, 4- and 6- weeks experiment) were compared with their respective NC and CC groups Table 1. Mn-SOD activity increased significantly (P<0.05) when copper-deficient groups ( 2-,4- and 6- weeks experiment) were compared with their respective NC, CC, and PF groups.

A similar pattern of increase was evident when pair-fed groups of the three experiments was compared with their respective NC and CC groups Table 1. Catalase activity increased significantly (P<0.05) when copper-deficient groups of 2-,4- and 6- weeks experiments were compared with their respective NC, CC and PF groups. A similar pattern of increase was observed when pair-fed groups of the three experiments was compared with their respective NC and CC groups Table 1.

DISCUSSION: Mammalian testes are highly sensitive to oxidative stress with consequent generation of reactive oxygen species due to disruption of cellular redox homeostasis. Non-enzymatic and enzymatic defense systems achieve cellular survival.

Wistar rat testes after a short duration of dietary copper deficiency caused a significant decrease in glutathione concentration in copper-deficient groups compared to respective negative and copper control groups. Glutathione, an intracellular reductant responsible for maintaining the enzymes' SH groups, is detected after its synthesis in mitochondria, endoplasmic reticulum nucleus, blood plasma, and bile ^{46, 47}.

A diminished level of glutathione indicates that cells are susceptible to oxidative stress and decreases both DNA and protein synthesis ⁴⁸. Moreover, it participates in the decomposition of H₂O_2, although the reaction is catalyzed by GSH peroxidase. Mitochondria are found plentiful in spermatids and spermatozoa and can generate reactive oxygen species under certain conditions. GSH efficiently scavenges free radicals, reactive oxygen species, and reactive nitrogen species ^{27, 33, 49} and its depletion in testes would enhance the vulnerability of spermatogenic cells to free radicals ⁵⁰.

High GSH level was observed at the onset of spermatogenesis ⁵¹, isolated spermatogenic cells in mice ⁵² with low GSH level in immature hamster Sertoli cells ⁵³. Agarwal and Vanha – Pertulla ⁵⁴ observed maximal GSH staining in the mid-piece as well as the tail region of spermatozoa.

GSH is important for sperm chromatin condensation following sperm penetration ⁵⁵ and significantly impacts motility pattern ⁵⁶. Ognik et al., ⁵⁷ observed a decline in GSH concentration in the brain and testes in copper-deficient Wistar rats accounting for the generation of free radicals. Human spermatozoa are highly susceptible to oxidative stress because of abundant polyunsaturated fatty acids and their capacity to generate reactive oxygen species ⁵⁸ and possess low concentration of antioxidant enzymes- catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase ⁵⁹ to neutralize ROS ⁶⁰. As observed in the study, deficiency may affect the spermatogenesis and its functions as well as the number of spermatozoa as cells with low GSH are prone to oxidative stress. Experiments revealed that the GSH effectively improves sperm morphology and motility ^{61, 62, 63, 64}. Moderate to low level of GSH has been reported in oligospermic, asthenozoo- and oligoastheno-zoospermia men ^{58, 65, 66, 67}.

The apparent decrement in pair-fed groups could be due to stress and starvation effect. Due to stress, the decline in GSH and free sulfhydryl groups have also been reported ^{33, 68}. Reactive oxygen species deleterious effects in mammalian testes are prevented mainly by extracellular SOD/SOD3, Cu/Zn-SOD/ SOD1, and Fe/Mn-SOD/ SOD2 ⁶⁹.

It was further reported that SOD3 secretion by Sertoli cells may be stimulated by germ cells through cytokines ⁶⁹. Cu/Zn SOD and Mn-SOD are expressed in Leydig, peritubular myoid, and Sertoli cells ⁷⁰. Experimental evidence indicated developmental and translational regulation of SOD‑2 mRNA levels with maximal levels of expression in early post‑meiotic germ cells ⁷¹. Authors ⁷² observed human spermatozoa are protected from increased mitochondrial superoxide radical generation by superoxide dismutase against the toxic effects of NADPH. The study reveals increased total SOD and Mn-SOD activities after copper deficiency which progressed as the duration increased, indicative of an imbalance of the redox state. Stress and starvation in the pair-fed group also exhibited an increased expression of the enzyme. A high level of total SOD and Mn-SOD in the present study can be associated with protecting the germ cells, Sertoli cells and Leydig cells against (i) enhanced superoxide radical (ii) increased generation of hydrogen peroxide and (iii) detrimental changes in seminiferous tubules after dietary copper deficiency.

Celino et al., ⁷³ showed a high level of SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Using the immunocytochemical technique, spermatogonia exhibited strong expression for Cu/Zn SOD and weak expression in advanced-stage germ cells.

Jow et al., ⁷⁴ observed the highest Cu/Zn SOD mRNA level in seminiferous tubules (stages VI-VIII) prior to spermiation. SOD -/- knock-out animals exhibited an increase in oxidative stress markers ⁷⁵ and reduced fertility rate in-vitro ⁷⁶. Garratt et al., ⁷⁷ reported impaired sperm motility and function impairment in Sod1-deficient mice. Dietary Cu restriction in weanling male rats revealed 68% decline in CuZnSOD activity, revealed 58% increase in superoxide anions, enhanced lipid peroxidation and damage to macromolecules ^{78, 79}.

Decreased activity of Cu/Zn superoxide dismutase (SOD1) indicates decreased copper stores ^{80, 81}. Copper deficient embryos exhibited malformation and low SOD activity, accounting for increased ROS generation ⁸². Ognik et al.,⁵⁷ observed low SOD activity in heart, lungs, and testes of copper-deficient male albino Wistar rats (Han IGS Rat [Crl: WI(Han)]) causing impairment of antioxidant defense system. The present study revealed decreased Cu-Zn SOD activity due to decreased availability of the Cu, which functions as a catalytic cofactor enhancing the vulnerability of germ cells, particularly spermatogonia being on the outside of testes barrier as well as testicular somatic cells to ROS. Catalase metabolizes hydrogen peroxide in peroxisomes; catalase bearing peroxisomes have been described in rodent Leydig cells ⁸³.

Neinicu et al.,⁸⁴ reported the presence of oxidases that are responsible for generating H₂O₂ in peroxisomes in Leydig, Sertoli, and peritubular cells, besides spermatogenesis, spermatocytes and round as well as elongated spermatids. Authors ^{70, 85, 86} reported no activity in primary spermatocytes and round spermatids. Ognik et al., ⁵⁷ reported high CAT activity in albino Wistar rats (Cu-0) testes of Wistar rats fed diet having 6.5 mg copper in the standard mineral mixture. The increased catalase activity in the present study could be due to the generation of H₂O₂ as a result of the direct reduction of O₂ to counteract the harmful effect of radicals reflecting enhanced ROS and oxidative stress, which would be deleterious gonads.

CONCLUSION: Dietary copper deficiency after 2-, 4- and 6- weeks caused dysregulation of glutathione enhanced the activity of total SOD, Mn-SOD and catalase while decreasing the expression of Cu-Zn SOD. This indicates that copper homeostasis was affected, which would have a deleterious effect on testes, causing dysfunction leading to infertility- the main cause of concern.

ACKNOWLEDGEMENT: Ms. Aastha Saini thanks the University Grants Commission for the award of Junior Research Fellowship (No. 22/06/2014 (1) EU-V) and Senior Fellowship. Ms. Ankita Rajendra Kurup thanks University Grants Commission, New Delhi, for the award of the Non NET fellowship. The authors gratefully acknowledge the Department of Zoology, Centre for Advanced Studies, University of Rajasthan, Jaipur, India, for providing necessary facilities.

CONFLICTS OF INTEREST: There are no conflicts of interest between authors.

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    How to cite this article:

    Saini A, Kurup AR and Nair N: Glutathione and antioxidant study in testes of Wistar rats after dietary copper deficiency. Int J Pharm Sci & Res 2022; 13(4): 1747-54. doi: 10.13040/IJPSR.0975-8232.13(4).1747-54.

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