Response of testicular antioxidant enzymes to hexachlorocyclohexane is species specific

Luna Samanta, G.B.N. Chainy

Asian J Androl 2002 Sep; 4: 191-194           

Keywords: hexachlorocyclohexane; rat; testes; lipid peroxidation; antioxidant defence

Abstract

Aim: To find out whether the response of testicular oxidative stress parameters to hexachlorocyclohexane (HCH) is species specific. Methods: In rats and mice (n=5 in each group), HCH was administered at a dose of 20 mgkg^-1day^-1 intraperitoneally for 30 days in 0.1 mL of refined groundnut oil. The control groups received equal volume of the vehicle. Animals were sacrificed 24 hours after the last injection and various oxidative stress parameters were measured immediately. Results: The level of both endogenous as well as FeSO₄ and ascorbic acid-stimulated lipid peroxidation was increased significantly in the HCH-treated rats, whereas the pattern was just the reverse in case of mice. Although the level of H₂O₂ content increased in response to HCH in both groups, a totally different trend was observed for the activity of the principal H₂O₂-metabolising enzyme, catalase. In case of rats, a significant decline in the activity of catalase was recorded in response to HCH whereas a sharp augmentation in the enzyme activity was noticed in mice. Similarly, the decreased activity of superoxide dismutase observed in rats remained unaltered in mice. Conclusion: HCH induces oxidative stress in the testis of both rats and mice. However, the pattern of response of testicular oxidative stress parameters seems to be species specific.

Hexachlorocyclohexane (HCH) is an organochlorine pesticide extensively used in agriculture and human and veterinary medicine [1]. By virtue of its lipophilicity, it is deposited in the lipid bilayer of all cell types following its entry into living system [1]. Testis is considered as one of the target organs of HCH toxicity owing to its high polyunsaturated fatty acid content. Involvement of reactive oxygen species (ROS) is postulated as one of the mechanisms through which most of the xenobiotics exert their noxious effects on the testis [2,3]. HCH is known to affect the testicular function in both rats [4] and mice [5]. A mechanism of HCH testicular toxicity seems to be the induction of oxidative stress in rat testis following acute and chronic treatment of the pesticide [6,7,8], which has been recently confirmed by Sujatha et al [9]. A direct inhibitory action of g-HCH on testicular steroidogenesis through reduction in the classical second messenger (cAMP) production has also been reported in cultured rat Leydig cells [10]. With this background, the present study was undertaken to find out whether there exists a generalized mechanism in HCH induced testicular dysfunction in mammals or the response is species speci-fic.

2 Materials and methods

Technical HCH was obtained from the Southern Pesticides Corporation Ltd. (India, CAS No. 58-89-8 and g-isomer content: 23.8 % by mass). All other chemicals used were of the highest purified grade com-mercially available.

2.2 Animals, pesticide treatment and biochemical estimations

Three months old, young, healthy and sexually mature male Swiss albino mice (25-30 g) and Wistar rats (200-300 g) were randomly divided into 2 groups each (n=5). Animals of Group II were administered technical HCH (20 mgkg^-1day^-1, intraperitoneally) suspended in 0.1 mL of refined groundnut oil for 30 days. Group I served as the control and received the vehicle only. Animals were sacrificed 24 h after the last injection and testes were quickly dissected out, washed free of blood with 0.9 % (w/v) ice-cold saline and processed immediately for estimation of protein, lipid peroxidation (LPX), the activity of superoxide dismutase (SOD; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6), and the levels of reduced glutathione (GSH) and hydrogen peroxide as described previously [6,11].

The susceptibility of testicular membrane lipids to in vitro peroxidation by extraneous burden of oxidants was assayed after 60 min of stimulation with 250 mM FeSO₄ and 10 mM ascorbic acid at 37. Pilot experiments have shown that under these conditions the peroxidation stimulation was the maximum [6]. Control samples were incubated under identical conditions without the oxidants (i.e. FeSO₄ and ascorbic acid).

Animal experiments were performed in accordance with the ethical standards provided by the Committee for the Purpose of Control and Supervision of Experi-ments on Animals (CPCSEA), Government of India.

Data are expressed as meanSD. Statistical analysis was performed using Students' t test. A difference was considered significant at P<0.05 level.

3 Results

The body weight and testicular weight did not change in response to HCH treatment in both animal groups, while the weights of accessory sex organs were reduced signifi-cantly (Table 1).

Table 1. Effect of HCH (20 mgkg^-1day^-1 for 30 days) on body weight (g) and weights of testis and accessory sex organs (mg/100 g body weight) in mice and rats. ^bP<0.05, compared with controls.

HCH treatment resulted in an increase in the level of lipid peroxidation (endogenous peroxidation) in the testi-cular crude homogenate by 79 % in rats. On the contrary, the same treatment schedule produced a 34 % decrease in the mice. In HCH treated rats, the in vitro LPX value in crude homogenate was significantly increased (by 34 %) after 30 days. On the other hand, in mice the level was significantly decreased (43 %). The level of H₂O₂ was increased in response to the pesticide in both groups. However, the magnitude of augmentation was much higher in rats (128 %) in comparison to mice (34 %). Total cytosolic SOD activity increased by 26 % in HCH treated rats whereas the pesticide failed to produce any effect in mice. In response to HCH, a 23 % reduction in CAT activity was noticed in rat testis while the enzyme activity was augmented by 12.5 % in mice testis. Testicular GSH content of rats was not affected by HCH treatment. On the contrary, its level was depleted by 25 % in mice testis (Table 2).

Table 2. Effect of HCH (20 mgkg^-1day^-1 for 30 days) on testicular oxidative stress parameters in mice and rats. meanSD, ^bP<0.05, compared with controls.

HCH is a mixture of steric isomers (i.e. a, b, g, d and e). The primary action of biologically active HCH isomers is the disruption of plasma membrane [12] that may affect cell survivability. Organochlorine pesticides including HCH are reported to reduce membrane fluidity [13]. The alterations in the fluidity of the membrane may be either due to a direct effect of the pesticides on struc-tural integrity of the membrane or an enhancement of membrane lipid peroxidation due to ROS generation [14]. The response of various biochemical components of tissues to HCH treatment differs from species to species. The levels of phospholipids and triglycerides are reported to be elevated in the liver of rats in response to HCH [15] whereas in mice, the level of triglycerides was found to be depleted without affecting the phospholipid composi-tion [16]. Since the phospholipid and fatty acid composition are the two important factors controlling tissue LPX [17], the observed elevation in the level of LPX in rat testicular crude homogenate and depletion in its level in mice could be due to differential alterations in their lipid profile in response to HCH treatment. In fact, Chowdhury et al [18] have reported accumulation of testicular lipid components along with fatty degeneration in testicular tissue of rat in response to g-HCH treatment for 45 days. A similar species specific response was also reported for the a-isomer of the pesticide where the pesticide elicited DNA strand breaks in primary cultures of rat and human, but not in mouse hepatocytes [19]. When comparative formation of chlorophenol metabolites were evaluated in HCH fed mouse and rat livers by HPLC, no significant species differences were noted in the formation of the metabolites studied both in vivo and in vitro [20]. The authors have opined that marked species difference that is observed in tumour induction between mice and rats by HCH may not be due to the different rate of formation of chlorophenol metabolite.

Antioxidant defence mechanism plays a pivotal role in protecting testes from the harmful effects of elevated ROS generation [21,22]. One of the important aspects of the antioxidant enzymes is their synergistic functioning. Any impairment in one member of the system will influence the activities of the other enzymes. A decrease in SOD activity in rat testis will favour the accumulation of superoxide radicals, which is known to inhibit CAT [23] , causing augmentation of H₂O₂ generation. Alter-natively, the possibility of H₂O₂ generation due to autoxi-dation of ascorbic acid by superoxide radicals [17] produced in response to HCH [24] can not be ruled out [7]. On the other hand, induction of CAT in mouse testis without any noticeable change in SOD activity needs further clarification. It is probable that the elevated H₂O₂ generation in response to the pesticide could be involved in the signal for CAT induction [25]. The decrease in GSH content in the testes of mice may possibly be either due to increase demand of the tripeptide for metabolism of lipid hydroperoxides by glutathione peroxidase (GPx) [26] or interaction of GSH with HCH-derived free radicals [27].

In the present study, the components of antioxidant defence studied are found to be differentially regulated in the animal groups. Puri and Kohli [28] have reported increase in hepatic content of xenobiotic metabolising enzymes, namely, cytochrome P₄₅₀ content and activities of NADPH cytochrome c reductase, aminopyrine N-demethylase, glutathione-s-transferase (GST) and haem oxygenase in rats in response to g-HCH. This was accompanied with induction of oxidative stress evidenced by elevated LPX level and depleted non-protein thiol content in the tissue. However, the pesticide did not evoke any response in rabbit and monkey. Similarly, an increase in the level of thiobarbituric acid reactive substances (index of LPX) and activities of SOD, CAT, GPx, GST and g-glutamyl transferase were accompanied by a reduction in the level of GSH in g-HCH poisoned patients[29]. In an earlier study from our laboratory, we have reported induction of oxidative stress in the liver of chick by HCH where an increase in the level of GSH and LPX in various hepatic subcellular fractions were noticed without any significant alteration in the activities of SOD and CAT [30]. Thus, it is apparent from the above discussion that the response of testicular oxidative stress parameters to HCH is species specific and the rats are more susceptible than the mice.

The authors are grateful to the Head of the Depart-ment of Zoology and International Centre for Applied Biotechnology and Policy Studies, Utkal University, Bhubaneswar 751 004, India for providing necessary laboratory facilities. Financial assistance to the Depart-ment under DRS programme and to GBNC under Research Award by University Grants Commission, New Delhi, India is gratefully acknowledged. LS acknowledges Senior Research Fellowship Extended by Council of Scientific and Industrial Research, New Delhi, India.

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Correspondence to: Prof. G.B.N. Chainy, Biochemistry Unit, Department of Zoology, Utkal University, Bhubaneswar-751004,Orissa, India.
Tel: +91-674-587 389 Fax: +91-674-509 107/509 037
E-mail: chainyg@sify.com, luna_samanta@rediffmail.com
Received 2002-03-05      Accepted 2002-06-27