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Oxidative stress enzymes are changed in opioid abusers and multidrug abusers
Journal of Clinical Neuroscience xxx (xxxx) xxx
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Neuropathological study
Oxidative stress enzymes are changed in opioid abusers and multidrug abusers Mitra-Sadat Sadat-Shirazi a, Mohammad-Reza Zarrindast a,b,c,⇑, Ghorbangol Ashabi d,⇑ a
Iranian National Center for Addiction Studies, Tehran University of Medical Sciences, Tehran, Iran Pharmacology Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran c Endocrinology and Metabolism Research Institute, Tehran University of Medical Science, Tehran, Iran d Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran b
a r t i c l e
i n f o
Article history: Received 30 September 2019 Accepted 31 December 2019 Available online xxxx Keywords: Postmortem Prefrontal cortex Malondialdehyde Super oxide dismutase, COX-2
a b s t r a c t The current study was designed to measure malondialdehyde level (MDA), super oxide dismutase (SOD) activity and COX-2 protein level in the prefrontal cortex (PFC) of drug-abusers. A total of 101 male drug abusers and 13 control subjects were gathered from the Iranian Legal Medicine center, Kahrizak, Tehran. Kind of death was determined by forensic pathologists, and the kind of drugs of abuse was detected using hair analysis. The medial prefrontal cortex (mPFC), lateral prefrontal cortex (lPFC), and orbitofrontal cortex (OFC) were dissected and were kept at 80 °C, until starting the assays. Our results indicated that the level of MDA was increased in the mPFC, lPFC and OFC of pure-opioid and multi-drug abusers compared with the control group. The SOD activity was reduced in the mPFC, lPFC and OFC of abusers in comparison to the control group. The protein level of COX-2 was decreased in the mPFC and lPFC of multi-drug abusers compared with the control group. This elevation in oxidative stress might be due to the increase of dopamine (as a consequence of drug abuse) or the direct effect of opioids and other drugs of abuse on oxidative agents. Antioxidant agents may be useful in preventing the damaging effect of oxidative agents in the brain of drug-addicted persons. Ó 2019 Published by Elsevier Ltd.
1. Introduction Drug dependence is one of the most important psychiatric disorder and health problems in the World, especially in the Middle East. Postmortem studies in this field might help us to detect new drugs to treated substance abuse disorder. Drugs could induce oxidative stress in two ways: (1) reduced the activity of the antioxidant system, and (2) increase the formation of free radicals. It might be possible that drugs activate oxidative stress in both mentioned ways [1]. It has been well described that morphine, methamphetamine and alcohol could increase oxidative stress [2–4]. Animal studies indicated that chronic morphine treatment reduced glutathione level in the liver and brain [5–7]. Also, it affects endogenous antioxidant defense such as superoxidase dismutase (SOD), catalase, and glutathione peroxidase [8]. Lipid peroxidation occurred when a free radical steal electron from lipids
⇑ Corresponding authors at: Department of Physiology, School of Medicine, Tehran University of Medical Sciences, P.O.box: 1417613151, Tehran, Iran. E-mail addresses: [email protected] (M.-S. Sadat-Shirazi), [email protected]. ir (M.-R. Zarrindast), [email protected] (G. Ashabi).
[9]. Since measuring lipid peroxidation in living subject is not possible, the level of malondialdehyde/thiobarbituric acid (MDA/TBA) reactive species in biological samples might be an excellent tool to evaluate lipid peroxidation level [10,11]. In addition to MDA, SOD has been considered as major biological markers of oxidative stress [12]. Among antioxidant enzymes SOD is the only known enzyme to directly scavenge a free radical. Oxidative stress is enhanced in many psychiatric disorders such schizophrenia [13–15], major depression disorder [16,17], bipolar disorder [18,19] and other psychiatric disorder [20]; however, there is a lack of studies in finding the relation between oxidative stress enzymes and substance abuse disorder. The oxidative stress has been indicated to be involved in generating mediators for inflammation. Cyclooxygenase-2 (COX-2) is an important neuro-inflammatory factors which could induce neuronal apoptosis and caspases cascade [21]. Moreover, COX-2 inhibitor is useful for treatment of cognitive dysfunction, analgesic drug deposition after morphineinduced analgesia in patients [22]. Postmortem studies are relevant and precise studies, the current investigation was designed to evaluate the MDA level, SOD activity and COX-2 protein level in the brain of humans who suffered from substance abuse. Since in Iran, morphine abuse
https://doi.org/10.1016/j.jocn.2019.12.064 0967-5868/Ó 2019 Published by Elsevier Ltd.
Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064
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(in the form of opium) is more prevalence, we chose pure opioid abusers. However; some of the abusers who consumed other drugs were categorized in multidrug abusers group. It should be noted all of our cases consumed drugs about seven years (according to family reports). Because of the important role of the PFC in addiction [23], three most important sub-region of the prefrontal cortex (orbitofrontal cortex (OFC), medial prefrontal cortex (mPFC), and lateral prefrontal cortex (lPFC)) were chosen for this study. 2. Materials and methods
an hour. Then, the membrane was incubated with primary antibody (COX-2, Abnova, Taiwan, 1/1000) overnight at 4 °C. In the second day, the membrane was washed 3 times (each time 10 min) with Tris buffer saline with tween 20 (TBST) and was transferred to the secondary antibody for an hour (Horseradish peroxidaselinked goat anti-rabbit IgG, Abcam, USA, 1:5000). Then, the membrane was washed again three times with TBST. Using Enhanced Chemiluminescence (ECL; Amersham, UK) Western blot detection system, the bonds were visualized in the radiographic film. We used Image J software for densitometry the bands and then the digits were analyzed by SPSS software.
2.1. Post-mortem brain tissue collection and subject characterization 2.5. Statistical analysis As described previously [24], total 101 male addict and 13 male control subjects were acquired from Iranian legal medicine organization, Kahrizak, Tehran, Iran. Kind of death and the kind of drugs abuse were determined by forensic pathologists and were described previously [24]. The mPFC (Brodmann’s area 9, 10, 12), lPFC (Brodmann’s area 10, 46) and OFC (Brodmann’s area 11, 12) were dissected according to the Paxinos atlas [25]. After dissecting, samples were immediately frozen in liquid nitrogen and then were kept at 80 °C. The kind of drug of abuse and chronic consumption were detected using hair analysis, as was described previously [24]. 2.2. Measurement of lipid peroxidation The MDA level was measured as an index of lipid peroxidation as was described by Placer et al. [26] which was based on detection of the color generated by the reaction of malondialdehyde and TBA. A 0.2 of PFC homogenate was maintained in 37 °C for an hour. A total of 30 ml of butylated hydroxytoluene was added to the homogenate, and 60 ml of Trichloroacetic acid also was added. The mixture was kept at 100 °C for 10 min and after that was centrifuged (2000 g, 4 °C, 10 min). The supernatant was mixed with TBA and was kept in 95 °C for an hour. The absorbance of the mixture was measured at 532 nm using a microplate reader (Awareness, USA) according to the blank solution. 2.3. Superoxide dismutase activity assay Briefly, SOD assay mixture contained 0.1 mL of supernatant, 1.2 mL of sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 mL of phenazine methosulphate (186 lM), 0.3 mL of nitroblue tetrazolium (300 lM), and 0.2 mL of NADH (750 lM) [27]. The reaction was happening by addition of 0.1 mL of NADH. After incubation at 30 °C for 90 s, the reaction was stopped by addition of 0.1 mL of glacial acetic acid. The reaction mixture was stirred with 4.0 mL of n-butanol. Color intensity of the chromogen in butanol was measured spectrophotometrically at 560 nm. One unit of enzyme activity was defined as the amount of enzyme which caused 50% inhibition of nitroblue tetrazolium reduction per mg of protein. 2.4. Protein extraction and western blotting The total protein was extracted using radioimmunoprecipitation assay buffer (RIPA buffer) and protease inhibitor as was described previously [28]. Thirty mg of protein was mixed with loading buffer (Tris, SDS, DTT, Glycerol, bromophenol blue) and was kept in 95 °C for 5 min. Then, samples were electrophoresis (Biorad, USA) in a polyacrylamide gel (10%). After finishing the electrophoresis, proteins were transferred to polyvinylidene fluoride (Chemicon Millipore Co. Temecula, USA) membrane. The membrane was incubated in skim milk (5%, Merck, Germany) for
Statistical Package for the Social Sciences (SPSS, version 21) was used to determine the changes between groups. One-Way analysis of variance (ANOVA) following post hoc test (Tukey) was used to determine the changes between groups. In addition, t-test analysis was used to determine changes between kinds of death (overdose and non-overdose). 3. Results 3.1. Demographic data Demographic data of abusers were presented previously [24]. However, Table 1 is shown that there is no statistical significance in age among groups. In addition, the age, total time of drug consumption, and kind of death (overdose and non-overdose) are shown in Table 1. 3.2. Lipid peroxidation was increased in the prefrontal cortex pureopioid and multi-drug abusers As is shown in Fig. 1-A, the level of malondialdehyde was increased in the mPFC of pure-opioid abusers and multi-drug abusers groups as compared with the control group (p < 0.05). The post-hoc Tukey test revealed that there was no significant difference between the MDA level in the mPFC of pure-opioid and multi-drug abusers. The changes in the MDA in the lPFC are shown in Fig. 1-A. The level of MDA was increased in the pure-opioid abusers and multi-drug abusers groups in comparison to the control (p < 0.05). In addition, MDA increased in the OFC of pure-opioid abusers and multi-drug abusers groups as compared with the control group (p < 0.01). 3.3. SOD activity was decreased in the prefrontal cortex pure-opioid and multi-drug abusers Fig. 1-B showed the level of SOD in the mPFC, lPFC and OFC of experimental groups. The level of SOD was reduced in the mPFC of pure-opioid abusers and multi-drug abusers groups as compared with the control group (p < 0.001). The level of SOD was decreased in the pure-opioid abusers and multi-drug abusers groups in comparison to the control in the lPFC (p < 0.01, p < 0.05, respectively). SOD activity was inhibited in the OFC of pure-opioid abusers and multi-drug abusers as compared with the control group (p < 0.05). 3.4. COX-2 protein level was elevated in the prefrontal cortex pureopioid and multi-drug abusers Fig. 2-A showed the represented blot of COX-2 protein level in the mPFC, lPFC and OFC of experimental groups. Densitometry analysis showed the level of COX-2 was increased in the mPFC of multi-drug abusers group as compared with the control group
Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064
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Table 1 Demographic data of subjects. The mean of age and duration of drug consumption (mean ± SEM) and the manner of death are presented in Table 1. There is no statistical significance in age between groups. Number
Control 13
Pure opioid abusers 56
Multi drug abusers 45
Total addict 101
Age (year) Duration of drug consumption (year) Cause of Death
36.308 ± 2.57 0 0 13 0
38.39 ± 1.14 8.6 ± 0.71 11 41 4
35.572 ± 1.44 6.93 ± 0.56 18 27 0
37.139 ± 0.91 7.83 ± 0.47 29 68 4
A
Overdose Non-overdose Unknown
4 3.5
Control *
Pure-opioid abusers #
*
Multi-drug abusers &
#
&&
MDA (nmol/mg)
3 2.5 2 1.5 1 0.5 0
Medial prefrontal cortex
B
7
Control
Lateral prefrontal cortex
Pure-opioid abusers
Orbitofrontal cortex
Multi-drug abusers
6
SOD (U/mg)
5 4 3
#
***
&
&
##
***
2 1 0
Medial prefrontal cortex
Lateral prefrontal cortex
Orbitofrontal cortex
Fig. 1. The level of MDA (nmol/mg) and SOD (U/mg). (A) MDA level in the mPFC of drug-abusers was increased compared with the control group (*p < 0.05). MDA level in the lPFC of drug-abusers was increased compared with the control group (#p < 0.05), and MDA level in the OFC of drug-abusers was increased compared with the control group (&p < 0.05, &&p < 0.01). (B) SOD level in the mPFC of drug-abusers was decreased compared with the control group (***p < 0.001). SOD level in the lPFC of drug-abusers was reduced compared with the control group (#p < 0.05, ##p < 0.01), and SOD level in the OFC of drug-abusers was reduced compared with the control group (& p < 0.05). Bars represent Mean ± S.D.
(p < 0.05). The protein level of COX-2 was amplified in the multidrug abusers group compared with the control in the lPFC (p < 0.01). COX-2 level was elevated in the OFC of both pureopioid abusers and multi-drug abusers as compared with the control group (p < 0.01).
4. Discussion The current results indicated that the level of MDA (as an index of lipid peroxidation) was increased in the PFC of pure-opioid and multidrug abusers. SOD, a strong superoxide scavenger enzyme, was reduced in the PFC of both pure-opioid and multi-drug abusers. In addition, COX-2 protein level was elevated in the mPFC
and lPFC for multi-drug abusers and it has been enhanced in the OFC of both pure-opioid and multidrug abusers. Three most important subregions of the prefrontal cortex are OFC, mPFC, and lPFC, which all of them have a vital role in cognitive function [23]. Emotion regulation, awareness, motivation, salience attribution, and decision making which are controlled by OFC and mPFC were altered in addiction [23]. Also, lPFC involved in self-control, motivation and drive, awareness, attention and flexibility, working memory, learning and memory, and decisionmaking, which all of these cognitive abilities impaired in the substance-abuse disorder [23]. In addition, lPFC has many connections with the premotor area that made this region as an important region in behavioral changes induced by drugs [29]. Previous data indicated that morphine administration in rat could increase oxidative stress in the cortex [6] and plasma [30].
Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064
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A COX-2
72kDa
β-actin
COX-2/β-actin(arbitrary unit)
B
3
46kDa
Control
Pure-opioid abusers
2.5
Multi-drug abusers &&
##
&&
* 2 1.5 1 0.5 0
Medial prefrontal cortex
Lateral prefrontal cortex
Orbitofrontal cortex
Fig. 2. Changing in protein level of COX-2 in the mPFC, lPFC, OFC of pure-opioid abusers and multi-drug abusers and control. (A) The blot with marker for COX-2 was represented, (B) densitometry and statistical analysis of COX-2 by Image J and SPSS software, respectively. COX-2 level in the mPFC of multi-drug abusers was increased compared with the control group (*p < 0.05). COX-2 level in the lPFC of multi-drug abusers was elevated compared with the control group (##p < 0.01), and COX-2 level in the OFC of drug-abusers (both POA and MDA) was reduced compared with the control group (&&p < 0.01). Bars represent Mean ± S.D.
It is well established that morphine enhanced dopamine level in the brain [31] and dopamine was oxidized by monoamine oxidase-B (MAO-B) [32]. The toxicity of metabolites derived from dopamine oxidization was described earlier [33–35]. One of the dopamine oxidation metabolites is hydrogen peroxide [36], which interacts with transition metal ion and produces hydroxyl radical [37]. However, morphine metabolites could directly induce superoxidase formation [38]. In addition, data revealed that opioid (like morphine, codeine, and methadone) generate reactive radical species [39]. One animal study revealed that chronic heroin treatment increased the MDA level in the brain and serum which might be related to the effect of heroin on serum antioxidant capacity and anti-oxidative enzyme activity in the brain [40]. In addition, morphine could increase oxidant factors in the hippocampus of morphine-treated rats [41]. We found that in addicted people, level of glutamate and N-methyl-D-aspartate (NMDA) started to increase in the brain [42]; COX-2 is generated by oxidative stress and also is activated predominantly by NMDA [43]. It has been speculated that COX-2 can induce NMDA neurotoxicity and finally mediate apoptosis in the neurons [44]. 5. Conclusions In conclusion, our data showed that oxidative stress was enhanced in the mPFC, OFC, and lPFC of pure-opioid and multidrug abusers which might be related to the direct effect of opioid on generating of reactive radical species or the effect of dopamine oxidation. One hypothesis is that oxidative stress and NMDA might activate COX-2 and subsequently increase neuronal inflammation and apoptosis. However; drugs which cause to block or reduce oxidative stress might be useful in treating chronic drug abusers. Author contributions Conception and design of research: Ashabi and Zarrindast; Performed experiments: Shirazi; Analyzed data: Shirazi; Interpreted
results of experiments: Ashabi and Zarrindast; Prepared figures: Ashabi and Zarrindast; Drafted manuscript: Ashabi and Zarrindast. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors acknowledge the grant support of the National Institute for Medical Research Development (NIMAD), Iran (Grant no. 963719). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jocn.2019.12.064. References [1] Sies H. Oxidative stress: a concept in redox biology and medicine. Redox Biol. 2015;4:180–3. [2] Skrabalova J et al. Morphine as a potential oxidative stress-causing agent. Mini Rev. Org. Chem. 2013;10(4):367–72. [3] Zou YJ et al. Apelin-13 protects PC12 cells from corticosterone-induced apoptosis through PI3K and ERKs activation. Neurochem. Res. 2016;41 (7):1635–44. [4] De Ternay J et al. Therapeutic prospects of cannabidiol for alcohol use disorder and alcohol-related damages on the liver and the brain. Front. Pharmacol. 2019;10. [5] Guzman DC et al. Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain. Neurochem. Res. 2006;31 (4):549–54. [6] Ozmen I et al. Spinal morphine administration reduces the fatty acid contents in spinal cord and brain by increasing oxidative stress. Neurochem. Res. 2007;32(1):19–25. [7] Sumathi T et al. Protective effect of Bacoside-A against morphine-induced oxidative stress in rats. Indian J. Pharm. Sci. 2011;73(4):409–15.
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Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Neuropathological study
Oxidative stress enzymes are changed in opioid abusers and multidrug abusers Mitra-Sadat Sadat-Shirazi a, Mohammad-Reza Zarrindast a,b,c,⇑, Ghorbangol Ashabi d,⇑ a
Iranian National Center for Addiction Studies, Tehran University of Medical Sciences, Tehran, Iran Pharmacology Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran c Endocrinology and Metabolism Research Institute, Tehran University of Medical Science, Tehran, Iran d Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran b
a r t i c l e
i n f o
Article history: Received 30 September 2019 Accepted 31 December 2019 Available online xxxx Keywords: Postmortem Prefrontal cortex Malondialdehyde Super oxide dismutase, COX-2
a b s t r a c t The current study was designed to measure malondialdehyde level (MDA), super oxide dismutase (SOD) activity and COX-2 protein level in the prefrontal cortex (PFC) of drug-abusers. A total of 101 male drug abusers and 13 control subjects were gathered from the Iranian Legal Medicine center, Kahrizak, Tehran. Kind of death was determined by forensic pathologists, and the kind of drugs of abuse was detected using hair analysis. The medial prefrontal cortex (mPFC), lateral prefrontal cortex (lPFC), and orbitofrontal cortex (OFC) were dissected and were kept at 80 °C, until starting the assays. Our results indicated that the level of MDA was increased in the mPFC, lPFC and OFC of pure-opioid and multi-drug abusers compared with the control group. The SOD activity was reduced in the mPFC, lPFC and OFC of abusers in comparison to the control group. The protein level of COX-2 was decreased in the mPFC and lPFC of multi-drug abusers compared with the control group. This elevation in oxidative stress might be due to the increase of dopamine (as a consequence of drug abuse) or the direct effect of opioids and other drugs of abuse on oxidative agents. Antioxidant agents may be useful in preventing the damaging effect of oxidative agents in the brain of drug-addicted persons. Ó 2019 Published by Elsevier Ltd.
1. Introduction Drug dependence is one of the most important psychiatric disorder and health problems in the World, especially in the Middle East. Postmortem studies in this field might help us to detect new drugs to treated substance abuse disorder. Drugs could induce oxidative stress in two ways: (1) reduced the activity of the antioxidant system, and (2) increase the formation of free radicals. It might be possible that drugs activate oxidative stress in both mentioned ways [1]. It has been well described that morphine, methamphetamine and alcohol could increase oxidative stress [2–4]. Animal studies indicated that chronic morphine treatment reduced glutathione level in the liver and brain [5–7]. Also, it affects endogenous antioxidant defense such as superoxidase dismutase (SOD), catalase, and glutathione peroxidase [8]. Lipid peroxidation occurred when a free radical steal electron from lipids
⇑ Corresponding authors at: Department of Physiology, School of Medicine, Tehran University of Medical Sciences, P.O.box: 1417613151, Tehran, Iran. E-mail addresses: [email protected] (M.-S. Sadat-Shirazi), [email protected]. ir (M.-R. Zarrindast), [email protected] (G. Ashabi).
[9]. Since measuring lipid peroxidation in living subject is not possible, the level of malondialdehyde/thiobarbituric acid (MDA/TBA) reactive species in biological samples might be an excellent tool to evaluate lipid peroxidation level [10,11]. In addition to MDA, SOD has been considered as major biological markers of oxidative stress [12]. Among antioxidant enzymes SOD is the only known enzyme to directly scavenge a free radical. Oxidative stress is enhanced in many psychiatric disorders such schizophrenia [13–15], major depression disorder [16,17], bipolar disorder [18,19] and other psychiatric disorder [20]; however, there is a lack of studies in finding the relation between oxidative stress enzymes and substance abuse disorder. The oxidative stress has been indicated to be involved in generating mediators for inflammation. Cyclooxygenase-2 (COX-2) is an important neuro-inflammatory factors which could induce neuronal apoptosis and caspases cascade [21]. Moreover, COX-2 inhibitor is useful for treatment of cognitive dysfunction, analgesic drug deposition after morphineinduced analgesia in patients [22]. Postmortem studies are relevant and precise studies, the current investigation was designed to evaluate the MDA level, SOD activity and COX-2 protein level in the brain of humans who suffered from substance abuse. Since in Iran, morphine abuse
https://doi.org/10.1016/j.jocn.2019.12.064 0967-5868/Ó 2019 Published by Elsevier Ltd.
Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064
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(in the form of opium) is more prevalence, we chose pure opioid abusers. However; some of the abusers who consumed other drugs were categorized in multidrug abusers group. It should be noted all of our cases consumed drugs about seven years (according to family reports). Because of the important role of the PFC in addiction [23], three most important sub-region of the prefrontal cortex (orbitofrontal cortex (OFC), medial prefrontal cortex (mPFC), and lateral prefrontal cortex (lPFC)) were chosen for this study. 2. Materials and methods
an hour. Then, the membrane was incubated with primary antibody (COX-2, Abnova, Taiwan, 1/1000) overnight at 4 °C. In the second day, the membrane was washed 3 times (each time 10 min) with Tris buffer saline with tween 20 (TBST) and was transferred to the secondary antibody for an hour (Horseradish peroxidaselinked goat anti-rabbit IgG, Abcam, USA, 1:5000). Then, the membrane was washed again three times with TBST. Using Enhanced Chemiluminescence (ECL; Amersham, UK) Western blot detection system, the bonds were visualized in the radiographic film. We used Image J software for densitometry the bands and then the digits were analyzed by SPSS software.
2.1. Post-mortem brain tissue collection and subject characterization 2.5. Statistical analysis As described previously [24], total 101 male addict and 13 male control subjects were acquired from Iranian legal medicine organization, Kahrizak, Tehran, Iran. Kind of death and the kind of drugs abuse were determined by forensic pathologists and were described previously [24]. The mPFC (Brodmann’s area 9, 10, 12), lPFC (Brodmann’s area 10, 46) and OFC (Brodmann’s area 11, 12) were dissected according to the Paxinos atlas [25]. After dissecting, samples were immediately frozen in liquid nitrogen and then were kept at 80 °C. The kind of drug of abuse and chronic consumption were detected using hair analysis, as was described previously [24]. 2.2. Measurement of lipid peroxidation The MDA level was measured as an index of lipid peroxidation as was described by Placer et al. [26] which was based on detection of the color generated by the reaction of malondialdehyde and TBA. A 0.2 of PFC homogenate was maintained in 37 °C for an hour. A total of 30 ml of butylated hydroxytoluene was added to the homogenate, and 60 ml of Trichloroacetic acid also was added. The mixture was kept at 100 °C for 10 min and after that was centrifuged (2000 g, 4 °C, 10 min). The supernatant was mixed with TBA and was kept in 95 °C for an hour. The absorbance of the mixture was measured at 532 nm using a microplate reader (Awareness, USA) according to the blank solution. 2.3. Superoxide dismutase activity assay Briefly, SOD assay mixture contained 0.1 mL of supernatant, 1.2 mL of sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 mL of phenazine methosulphate (186 lM), 0.3 mL of nitroblue tetrazolium (300 lM), and 0.2 mL of NADH (750 lM) [27]. The reaction was happening by addition of 0.1 mL of NADH. After incubation at 30 °C for 90 s, the reaction was stopped by addition of 0.1 mL of glacial acetic acid. The reaction mixture was stirred with 4.0 mL of n-butanol. Color intensity of the chromogen in butanol was measured spectrophotometrically at 560 nm. One unit of enzyme activity was defined as the amount of enzyme which caused 50% inhibition of nitroblue tetrazolium reduction per mg of protein. 2.4. Protein extraction and western blotting The total protein was extracted using radioimmunoprecipitation assay buffer (RIPA buffer) and protease inhibitor as was described previously [28]. Thirty mg of protein was mixed with loading buffer (Tris, SDS, DTT, Glycerol, bromophenol blue) and was kept in 95 °C for 5 min. Then, samples were electrophoresis (Biorad, USA) in a polyacrylamide gel (10%). After finishing the electrophoresis, proteins were transferred to polyvinylidene fluoride (Chemicon Millipore Co. Temecula, USA) membrane. The membrane was incubated in skim milk (5%, Merck, Germany) for
Statistical Package for the Social Sciences (SPSS, version 21) was used to determine the changes between groups. One-Way analysis of variance (ANOVA) following post hoc test (Tukey) was used to determine the changes between groups. In addition, t-test analysis was used to determine changes between kinds of death (overdose and non-overdose). 3. Results 3.1. Demographic data Demographic data of abusers were presented previously [24]. However, Table 1 is shown that there is no statistical significance in age among groups. In addition, the age, total time of drug consumption, and kind of death (overdose and non-overdose) are shown in Table 1. 3.2. Lipid peroxidation was increased in the prefrontal cortex pureopioid and multi-drug abusers As is shown in Fig. 1-A, the level of malondialdehyde was increased in the mPFC of pure-opioid abusers and multi-drug abusers groups as compared with the control group (p < 0.05). The post-hoc Tukey test revealed that there was no significant difference between the MDA level in the mPFC of pure-opioid and multi-drug abusers. The changes in the MDA in the lPFC are shown in Fig. 1-A. The level of MDA was increased in the pure-opioid abusers and multi-drug abusers groups in comparison to the control (p < 0.05). In addition, MDA increased in the OFC of pure-opioid abusers and multi-drug abusers groups as compared with the control group (p < 0.01). 3.3. SOD activity was decreased in the prefrontal cortex pure-opioid and multi-drug abusers Fig. 1-B showed the level of SOD in the mPFC, lPFC and OFC of experimental groups. The level of SOD was reduced in the mPFC of pure-opioid abusers and multi-drug abusers groups as compared with the control group (p < 0.001). The level of SOD was decreased in the pure-opioid abusers and multi-drug abusers groups in comparison to the control in the lPFC (p < 0.01, p < 0.05, respectively). SOD activity was inhibited in the OFC of pure-opioid abusers and multi-drug abusers as compared with the control group (p < 0.05). 3.4. COX-2 protein level was elevated in the prefrontal cortex pureopioid and multi-drug abusers Fig. 2-A showed the represented blot of COX-2 protein level in the mPFC, lPFC and OFC of experimental groups. Densitometry analysis showed the level of COX-2 was increased in the mPFC of multi-drug abusers group as compared with the control group
Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064
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Table 1 Demographic data of subjects. The mean of age and duration of drug consumption (mean ± SEM) and the manner of death are presented in Table 1. There is no statistical significance in age between groups. Number
Control 13
Pure opioid abusers 56
Multi drug abusers 45
Total addict 101
Age (year) Duration of drug consumption (year) Cause of Death
36.308 ± 2.57 0 0 13 0
38.39 ± 1.14 8.6 ± 0.71 11 41 4
35.572 ± 1.44 6.93 ± 0.56 18 27 0
37.139 ± 0.91 7.83 ± 0.47 29 68 4
A
Overdose Non-overdose Unknown
4 3.5
Control *
Pure-opioid abusers #
*
Multi-drug abusers &
#
&&
MDA (nmol/mg)
3 2.5 2 1.5 1 0.5 0
Medial prefrontal cortex
B
7
Control
Lateral prefrontal cortex
Pure-opioid abusers
Orbitofrontal cortex
Multi-drug abusers
6
SOD (U/mg)
5 4 3
#
***
&
&
##
***
2 1 0
Medial prefrontal cortex
Lateral prefrontal cortex
Orbitofrontal cortex
Fig. 1. The level of MDA (nmol/mg) and SOD (U/mg). (A) MDA level in the mPFC of drug-abusers was increased compared with the control group (*p < 0.05). MDA level in the lPFC of drug-abusers was increased compared with the control group (#p < 0.05), and MDA level in the OFC of drug-abusers was increased compared with the control group (&p < 0.05, &&p < 0.01). (B) SOD level in the mPFC of drug-abusers was decreased compared with the control group (***p < 0.001). SOD level in the lPFC of drug-abusers was reduced compared with the control group (#p < 0.05, ##p < 0.01), and SOD level in the OFC of drug-abusers was reduced compared with the control group (& p < 0.05). Bars represent Mean ± S.D.
(p < 0.05). The protein level of COX-2 was amplified in the multidrug abusers group compared with the control in the lPFC (p < 0.01). COX-2 level was elevated in the OFC of both pureopioid abusers and multi-drug abusers as compared with the control group (p < 0.01).
4. Discussion The current results indicated that the level of MDA (as an index of lipid peroxidation) was increased in the PFC of pure-opioid and multidrug abusers. SOD, a strong superoxide scavenger enzyme, was reduced in the PFC of both pure-opioid and multi-drug abusers. In addition, COX-2 protein level was elevated in the mPFC
and lPFC for multi-drug abusers and it has been enhanced in the OFC of both pure-opioid and multidrug abusers. Three most important subregions of the prefrontal cortex are OFC, mPFC, and lPFC, which all of them have a vital role in cognitive function [23]. Emotion regulation, awareness, motivation, salience attribution, and decision making which are controlled by OFC and mPFC were altered in addiction [23]. Also, lPFC involved in self-control, motivation and drive, awareness, attention and flexibility, working memory, learning and memory, and decisionmaking, which all of these cognitive abilities impaired in the substance-abuse disorder [23]. In addition, lPFC has many connections with the premotor area that made this region as an important region in behavioral changes induced by drugs [29]. Previous data indicated that morphine administration in rat could increase oxidative stress in the cortex [6] and plasma [30].
Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064
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M.-S. Sadat-Shirazi et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx
A COX-2
72kDa
β-actin
COX-2/β-actin(arbitrary unit)
B
3
46kDa
Control
Pure-opioid abusers
2.5
Multi-drug abusers &&
##
&&
* 2 1.5 1 0.5 0
Medial prefrontal cortex
Lateral prefrontal cortex
Orbitofrontal cortex
Fig. 2. Changing in protein level of COX-2 in the mPFC, lPFC, OFC of pure-opioid abusers and multi-drug abusers and control. (A) The blot with marker for COX-2 was represented, (B) densitometry and statistical analysis of COX-2 by Image J and SPSS software, respectively. COX-2 level in the mPFC of multi-drug abusers was increased compared with the control group (*p < 0.05). COX-2 level in the lPFC of multi-drug abusers was elevated compared with the control group (##p < 0.01), and COX-2 level in the OFC of drug-abusers (both POA and MDA) was reduced compared with the control group (&&p < 0.01). Bars represent Mean ± S.D.
It is well established that morphine enhanced dopamine level in the brain [31] and dopamine was oxidized by monoamine oxidase-B (MAO-B) [32]. The toxicity of metabolites derived from dopamine oxidization was described earlier [33–35]. One of the dopamine oxidation metabolites is hydrogen peroxide [36], which interacts with transition metal ion and produces hydroxyl radical [37]. However, morphine metabolites could directly induce superoxidase formation [38]. In addition, data revealed that opioid (like morphine, codeine, and methadone) generate reactive radical species [39]. One animal study revealed that chronic heroin treatment increased the MDA level in the brain and serum which might be related to the effect of heroin on serum antioxidant capacity and anti-oxidative enzyme activity in the brain [40]. In addition, morphine could increase oxidant factors in the hippocampus of morphine-treated rats [41]. We found that in addicted people, level of glutamate and N-methyl-D-aspartate (NMDA) started to increase in the brain [42]; COX-2 is generated by oxidative stress and also is activated predominantly by NMDA [43]. It has been speculated that COX-2 can induce NMDA neurotoxicity and finally mediate apoptosis in the neurons [44]. 5. Conclusions In conclusion, our data showed that oxidative stress was enhanced in the mPFC, OFC, and lPFC of pure-opioid and multidrug abusers which might be related to the direct effect of opioid on generating of reactive radical species or the effect of dopamine oxidation. One hypothesis is that oxidative stress and NMDA might activate COX-2 and subsequently increase neuronal inflammation and apoptosis. However; drugs which cause to block or reduce oxidative stress might be useful in treating chronic drug abusers. Author contributions Conception and design of research: Ashabi and Zarrindast; Performed experiments: Shirazi; Analyzed data: Shirazi; Interpreted
results of experiments: Ashabi and Zarrindast; Prepared figures: Ashabi and Zarrindast; Drafted manuscript: Ashabi and Zarrindast. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors acknowledge the grant support of the National Institute for Medical Research Development (NIMAD), Iran (Grant no. 963719). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jocn.2019.12.064. References [1] Sies H. Oxidative stress: a concept in redox biology and medicine. Redox Biol. 2015;4:180–3. [2] Skrabalova J et al. Morphine as a potential oxidative stress-causing agent. Mini Rev. Org. Chem. 2013;10(4):367–72. [3] Zou YJ et al. Apelin-13 protects PC12 cells from corticosterone-induced apoptosis through PI3K and ERKs activation. Neurochem. Res. 2016;41 (7):1635–44. [4] De Ternay J et al. Therapeutic prospects of cannabidiol for alcohol use disorder and alcohol-related damages on the liver and the brain. Front. Pharmacol. 2019;10. [5] Guzman DC et al. Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain. Neurochem. Res. 2006;31 (4):549–54. [6] Ozmen I et al. Spinal morphine administration reduces the fatty acid contents in spinal cord and brain by increasing oxidative stress. Neurochem. Res. 2007;32(1):19–25. [7] Sumathi T et al. Protective effect of Bacoside-A against morphine-induced oxidative stress in rats. Indian J. Pharm. Sci. 2011;73(4):409–15.
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Please cite this article as: M.-S. Sadat-Shirazi, M. R. Zarrindast and G. Ashabi, Oxidative stress enzymes are changed in opioid abusers and multidrug abusers, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.12.064