Journal of Steroid Biochemistry & Molecular Biology 75 (2000) 299 – 306 Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis Tatjana S. Kostic, Silvana A. Andric, Desanka Maric, Radmila Z. Kovacevic * Institute of Biology, Faculty of Sciences, Uniersity of Noi Sad, Dositeja Obradoica Square 2, 21000 Noi Sad, Serbia, Yugoslaia Accepted 11 October 2000 Abstract The messenger role of nitric oxide (NO) in immobilization stress-induced inhibition of testicular steroidogenesis has been previously suggested. In accord with this, here, we show that the intratesticular injection of isosorbide dinitrate (ISDN; 2 ×2.5 mg/testis), an NO donor, mimicked the action of stress on serum testosterone concentrations and hCG-stimulated testosterone production in rat testicular tissue. When added in vitro, ISDN inhibited testicular 3-hydroxysteroid dehydrogenase and 17-hydroxylase/lyase. Immobilization stress and injections of ISDN also decreased the activity of catalase, glutathione peroxidase, glutathione transferase, and glutathione reductase in the interstitial compartment of testis. When stressed rats were treated concomitantly with bilateral intratesticular injections of N  -nitro-L-arginine methyl ester, a non-selective NOS inhibitor (2 ×600 g/testis), the activities of antioxidative enzymes, as well as serum testosterone concentration, were partially normalized. These results indicate that stress-induced stimulation of the testicular NO signalling pathway leads to inhibition of both steroidogenic and antioxidant enzymes. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Antioxidant enzymes; Nitric oxide; Steroidogenesis; Stress; Testosterone www.elsevier.com/locate/jsbmb 1. Introduction Nitric oxide (NO) is produced by testicular tissues and acts as an autocrine/paracrine messenger in local regulation of steroidogenesis [1]. Several lines of evi- dence have also suggested that NO mediates the stress- induced downregulation of testicular steroidogenesis. For example, in-vitro androgenesis by decapsulated testes from stressed rats exposed to human chorionic gonadotrophin (hCG) was significantly reduced. This inhibition was accompanied by an elevation in the testicular nitrite, a stable oxidation product of NO pathway [2,3]. The impaired testicular steroidogenesis in stressed rats coincided with a significant inhibition of 3-hydroxysteroid dehydrogenase (3-HSD) [4,5], 17- hydroxylase/lyase (P450c17) [5 – 7], and NADPH-P450 reductase [8]. In addition to steroidogenic enzymes, the rise in intratesticular NO concentration may affect the activity of some antioxidant enzymes, leading to an imbalance of the prooxidant versus antioxidant status in the inter- stitial tissue and an increase in the production of reac- tive oxygen species. Namely, it is shown that NO donors, S -nitroso-N -acetylpenicillamine (SNAP) and sodium nitropruside (SNP), inhibited catalase activity in a concentration-dependent manner [9]. Furthermore, SNAP inhibited bovine glutathione peroxidase in a dose- and time-dependent manner [10]. It is also possi- ble that inhibition of P450-dependent enzymes is ac- companied by facilitation of the prooxidant status in the interstitial tissue and therefore by an additional inhibition of the antioxidant enzyme system in this compartment of the testis [11,12]. If the activity of the antioxidative enzymes is impaired in stress rats, this may result in facilitation of lipid peroxidation, which in turn could further inhibit the activity of the steroidogenic membrane-bound enzymes [11,12]. Thus, it is reasonable to speculate that the antigonadal effects of acute stress result from a dual inhibitory action of NO signalling pathway, on steroidogenic and antioxi- dant enzymes. * Corresponding author. Tel.: +381-21-58988; fax: +381-21- 450620. E-mail address: radmilak@unsim.ns.ac.yu (R.Z. Kovacevic). 0960-0760/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S0960-0760(00)00185-0