Protective effect of zinc sulfate and continuous/interval training on liver oxidative stress in morphine-withdrawal syndrome in rats

Introduction: In this study, the effect of zinc sulfate (ZS) supplement and eight-week continuous training (CT) and interval training (IT) on liver oxidative stress of morphine-dependent rats following withdrawal syndrome (WS) were evaluated. Methods: Seventy Wistar rats were randomly divided into seven groups: control rats, withdrawing rats (WS), withdrawing rats receiving 9mg/kg ZS orally (WS+Z), withdrawing rats under CT (WS+CT), withdrawing rats under IT (WS+IT), withdrawing rats under CT and receiving 9mg/kg zinc sulfate (WS+Z+CT), withdrawing rats under IT and receiving 9mg/kg zinc sulfate (WS+Z+IT). Animals were addicted by 0.4g/l morphine sulfate in 21 days. Animals in the training groups ran on a treadmill and received ZS 5 days/week for 8 weeks. At the end of the study, oxidative stress in liver tissue and liver enzymes were measured by spectrophotometric and ELISA methods. Results: ZS supplement, CT/IT led to decrease DNA damage and malondialdehyde in comparison with morphine group. Also, ZS, CT and IT significantly elevated levels in superoxide dismutase, catalase activity, total antioxidant capacity and thiol groups in the liver of rats in comparison with morphine group. Additionally, it is observed that ZS and CT/IT made a significant reduction in aspartate aminotransferase levels in comparison to the morphine group. Conclusion: CT/IT with ZS because of its antioxidant effects has the potential to be used for decreased withdrawal syndrome complications. http://dx.doi. org/10.32598/ppj.24. 4.10 iD D ow nl oa de d fr om p pj . p hy ph a. ir at 6 :4 0 + 04 30 o n W ed ne sd ay A pr il 21 st 2 02 1 [ D O I: 10 . 3 25 98 /p pj . 2 4. 4. 10 ] 277 | Physiol Pharmacol 24 (2020) 276-284 Hasani et al. Opioids continued to cause the most harm, accounting for 76% of the deaths where drug use disorders were implicated (Liu et al., 1999) . Morphine, the main component of opium, is perhaps the oldest drug known to man. Morphine decreases stimulation of pain nervous path neurons by connecting to μ-type receptors. Therefore, it is used as a strong anti-pain largely to relieve severe pain (Gordon et al., 1995). Nevertheless, long-term using of these drugs exerts several adverse side-effects, including addiction, tolerance, respiratory depression, immunosuppression and constipation. Addiction is one of the most important issues in human societies, which not only makes social and behavioral disorders but also impose great financial influencing (Schütz et al., 2018). Liver is one of the vital body organs that plays a major role in detoxification of the toxic elements and chemical drugs. Liver is considered as a major tissue of morphine biotransformation where morphine exerts several adverse toxic, immunological and oncogenic effects (Salahshoor et al., 2016). Increased liver enzymes including aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) are reported in laboratory animals after receiving morphine (Salahshoor et al., 2018). Oxidative stress plays a crucial role in toxic liver damage (Todaka et al., 2005). Studies showed that morphine results in oxidative stress by inducing the generation of reactive oxygen species (Gach et al., 2011). Samarghandian et al. (2014) reported that morphine consumption increased induction of lipid peroxidation and decreased anti-oxidant system’s power (the activity of enzymes such as superoxide dismutase [SOD] and catalase [CAT]) which is associated with liver toxicity and damage. Studies showed that morphine is converted to morphinone by morphine-6-dehydrogenase, and after conjugate with glutathione, and removed, which reduces glutathione sources (Nagamatsu et al., 1986). The antioxidants can protect the liver tissue against oxidative damage. Exercise plays a major role in human health. The beneficial effects of regular exercise and physical activity have been known for a long time. Exercise is part of the treatment of many diseases via induces the antioxidant defense’s in several tissues such as liver, heart and kidney (Simioni et al., 2018). In addition, exercise and physical activity have caused increased blood circulation in the liver and accelerated biotransformation and they also decreased liver oxidative toxic stress (Habib et al., 2020; Trefts et al., 2015). Trace element homeostasis is critical for antioxidant defense systems. Zinc incorporates in the function of more than 300 enzymes. Studies have shown that zinc plays a specific role in biochemical processes such as cell respiration, DNA propagation, maintaining the integrity of cell membrane and reducing free radicals (Stefanidou et al., 2006). Zinc ions defenses against free radicals by competitive role against copper and iron ions that induced producing free radicals during Haber Weiss reaction. Also, animal-based studies have shown that zinc deficiency can make a significant reduction in vitamin E serum levels. Vitamin E is a protective agent against oxidative damage and prohibits lipoproteins oxidation (Eide, 2011). Zinc deficiency well-documented in opioid consumers (Ciubotariu et al., 2015). However, based on our studies, there is little information regarding the effect of exercise or zinc supplementation on oxidative stress markers in morphine-dependent rats following withdrawal syndrome. Hence, investigating the effect of exercise and zinc supplementation on liver oxidative stress in the opium users could be considerably beneficial for withdrawing the opium. Therefore, in the current study, we assessed the impact of interval/continuous exercise and zinc supplementation on the liver oxidative stress in morphine-dependent rats following withdrawal syndrome. Materials and methods In this experimental study, 70 male Wistar rats (250±20g) were used. Animals were maintained in the animal house with free access to water and food throughout 12:12 hour’s light/dark cycle. All ethical considerations were confirmed by the given instructions of Hamadan University of Medical Sciences (IR.UMSHA.REC.1395.195). Animals were randomly categorized into seven groups (n=10): Group 1: healthy control rats (C); Group 2: withdrawing rats without exercise training and receiving normal saline (WS); Group 3: withdrawing rats receiving 9mg/kg zinc sulfate orally (WS+ZS); Group 4: withdrawing rats under continuous exercise training (WS+CT); Group 5: withdrawing rats under D ow nl oa de d fr om p pj . p hy ph a. ir at 6 :4 0 + 04 30 o n W ed ne sd ay A pr il 21 st 2 02 1 [ D O I: 10 . 3 25 98 /p pj . 2 4. 4. 10 ] Effect of training and zinc sulfate on addicted rats Physiol Pharmacol 24 (2020) 276-284 | 278 interval exercise training (WS+IT); Group 6: withdrawing rats under continuous exercise training receiving 9mg/kg zinc sulfate (WS+ZS+CT); Group 7: withdrawing rats under interval exercise training receiving 9mg/kg zinc sulfate (WS+ZS+IT). Addiction Animals in morphine-dependent groups were addicted by 0.4g/l morphine sulfate during 21 days. In addition, sucrose (40mg/m) was added to drinking water due to the bitter taste of morphine. To ensure that the morphine-induced dependency in the animals, 1-2 rats in each working group received 1mg/kg naloxone (Sigma-Aldrich, St. Louis, MO, USA, IP) (Zarrinkalam et al., 2016). After being assured of morphine, they were put on the treadmill for 1 week to get acquainted with the treadmill for 15 minutes at a speed of 13m/s. Rats that did so easily were selected as the study groups. Then animals of the training groups performed continuous/interval exercise training and 9mg/kg zinc sulfate were orally for five days in a week for 8 weeks. Continuous training In this protocol, rats were exercised for 8 weeks, 5 days/week. Rats had performed the training with 12m/min for 16 minutes. During 8 weeks, the training speed and duration were gradually increased and they exercised for 51 minutes with 18m/min on the treadmill (Table 1).