Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Letter to the Editor Association between null alleles of GSTM1 and GSTT1 and dependence to heroin and opium To the Editors: Opiates may cause oxidative stress in drug dependent persons (Ghazavi et al., 2013; Soykut et al., 2013). Glutathione S-transferases (GSTs) play an important role in cellular detoxification of xenobiotics. Several GST gene families, including mu and theta, have been identified. The null alleles of genes encoding GSTM1 (a member of class mu; OMIM: 138350) and GSTT1 (a member of class theta; OMIM: 600436) have been well defined in human. In has been reported that GSTT1 and GSTM1 are expressed in the brain (Juronen et al., 1996). Although the association between GSTs polymorphisms and susceptibility to methamphetamine abuse has been reported (Nakatome et al., 2009; Khalighinasab et al., 2015), there are no study investigating the association between these genetic variations and risk of heroin dependence (HD) and opium dependence (OD). Therefore, the present study was carried out. This report consists of two case–control studies performed in Shiraz (southern Iran). In the first study, 442 HD subjects (42 females, 400 males) and 799 healthy controls (137 females, 662 males) were included. A detailed description of these subjects has been reported in our previous report (Saify et al., 2014). In the second study, 143 OD (12 females, 131 males) and 570 healthy controls (55 females, 515 males) were included. All patients were assessed using the Structured Clinical Interview based on Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria for heroine and opium dependence. Moreover, urine drug screens were obtained. All patients were interviewed by a senior psychiatrist. The patients were in methadone maintenance for treating OD and all of them reported opium as their primary drug of choice. Control individuals were blood donors, who declared that they did not suffer from substance abuse. The mean age (S.D.) of the patients and the controls were 39.6 (11.3) and 40.8 (10.7) years, respectively. There was no significant difference in the gender and age distribution between the patient and control groups (P 40.05). The participants were selected from Persian Muslims (Caucasians) living in Shiraz (Fars province, southern Iran). This study was approved by the Shiraz University ethics committee and the informed consent was obtained from each subject. Using the QUANTO (http://biostats.usc.edu/software) software, to detect a real difference in genotypic frequency with a power of 0.80, α ¼ 0.05, OR ¼1.50, and 50% frequency of the GSTM1 null genotype; a minimum sample of 390 would be necessary. With similar assumptions and 25% frequency of the GSTT1 null genotype; a minimum sample of 560 would be necessary. The present case–control studies are more than sufficiently powered to detect a small–medium effect in genotype frequency between cases and their control groups. The PCR conditions for determining the genotypes of GSTT1 and GSTM1 and quality control were previously reported (Khalighinasab et al., 2015). http://dx.doi.org/10.1016/j.psychres.2015.05.004 0165-1781/& 2015 Elsevier Ireland Ltd. All rights reserved.
Table 1 shows that the null genotypes of GSTM1 (OR¼ 0.90, 95% CI: 0.71–1.14, P ¼0.401) and GSTT1 (OR¼ 1.25, 95% CI: 0.95–1.65, P¼ 0.107) were not associated with the risk of HD. After stratification of the participants according to their genders, the same associations were observed. The present study revealed that the GSTM1-null genotype was not associated with the susceptibility to OD (OR¼ 0.69, 95% CI: 0.48–1.00, P ¼0.052). However, the null genotype of GSTT1 was significantly associated with OD (OR ¼1.56, 95% CI: 1.02–2.39, P¼ 0.037) (Table 1). To investigate whether the combinations of the genotypes could alter the risk of substance abuse, we considered the associations between combinations of the genotypes and risks of HD and OD. The reference group consisted of individuals with “null genotype of GSTM1/positive genotype of GSTT1” (Table 1). Statistical analysis revealed that the combination of “positive genotype of GSTM1/null genotype of GSTT1” significantly associated with either HD (OR ¼1.52, 95% CI: 1.03–2.25, P ¼0.034) or OD (OR ¼2.60, 95% CI: 1.44–4.69, P¼ 0.001). Previous studies indicated that heroin and opium dependent persons are under oxidative stress conditions (Ghazavi et al., 2013; Soykut et al., 2013). We know that oxidative stress may occur due to increase in the generation of free radicals or in reduction in the level of antioxidant enzymes. Oxidative stress plays an important role in the pathogenesis of different diseases, especially in the Table 1 Associations between GSTM1 and GSTT1 polymorphisms and dependence to heroin and opium. Genotypes
Controls
Patients
Heroin dependent group GSTM1 polymorphism Positive 367 214 Null 432 228 GSTT1 polymorphism Positive 632 332 Null 167 110 GSTM1/GSTT1 combination genotypes Null/positive 338 176 Positive/positive 294 156 Positive/null 73 58 Null/null 94 52 Opium dependent group GSTM1 polymorphism Positive 255 77 Null 315 66 GSTT1 polymorphism Positive 460 104 Null 110 39 GSTM1/GSTT1 combination genotypes Null/positive 249 50 Positive/positive 211 54 Positive/null 44 23 Null/null 66 16
OR
95% CI
P
1.0 0.90
– 0.71–1.14
– 0.401
1.0 1.25
– 0.95–1.65
– 0.107
1.0 1.01 1.52 1.06
– 0.78–1.33 1.03–2.25 0.72–1.56
– 0.890 0.034 0.758
1.0 0.69
– 0.48–1.00
– 0.052
1.0 1.56
– 1.02–2.39
– 0.037
1.0 1.27 2.60 1.20
– 0.83–1.95 1.44–4.69 0.64–2.25
– 0.265 0.001 0.555
Letter to the Editor / Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
brain. It leads to DNA and cell damage and alter the Ca2 þ channel function (Hool, 2007). Our present findings indicate that the null genotype of GSTT1 (but not the GSTM1 null genotype) is significantly associated with elevation of the risk of heroine and opium dependence. It should be mentioned that at the present time, it is premature to discuss the cause and effect of the GSTM1 and GSTT1 null alleles on the dependency of heroin and opium. Determination of GSTM1 and GSTT1 gene expression levels of cells treated with these drugs are recommended for future experiments. Population stratification and use of blood donors as control group without any verification of whether or not they met criteria for a substance use disorder are limitations of the present study.
Nakatome, M., Miyaji, A., Mochizuki, K., et al., 2009. Association between the GST genetic polymorphisms and methamphetamine abusers in the Japanese population. Legal Medicine 11, 468–470. Saify, K., Saadat, I., Saadat, M., 2014. Association between VNTR polymorphism in promoter region of prodynorphin (PDYN) gene and heroin dependence. Psychiatry Research 219, 690–692. Soykut, B., Eken, A., Erdem, O., et al., 2013. Oxidative stress enzyme status and frequency of micronuclei in heroin addicts in Turkey. Toxicology Mechanisms and Methods 23, 684–688.
Mohammad Rashid Khalighinasab, Khyber Saify, Mostafa Saadat n Department of Biology, College of Sciences, Shiraz University, Shiraz 71454, Iran E-mail address: [email protected] (M. Saadat)
Conflict of interest
Received 18 November 2014 15 April 2015 2 May 2015
No competing interests are declared by any of the authors. Acknowledgments The authors are indebted to the participants for their close cooperation. The authors are indebted to Dr. Raheleh Masoudi for critical reading of the manuscript. This study was supported by Shiraz University (93GCU1M1741). References Ghazavi, A., Mosayebi, G., Solhi, H., et al., 2013. Serum markers of inflammation and oxidative stress in chronic opium (Taryak) smokers. Immunology Letters 153, 22–26. Hool, L.C., 2007. Evidence for the regulation of L-type Ca2 þ channels in the heart by reactive oxygen species – mechanism for mediating pathology. Proceedings of the Australian Physiological Society 38, 87–93. Juronen, E., Tasa, G., Uuskula, M., et al., 1996. Purification, characterization and tissue distribution of human class theta glutathione S-transferase T1-1. Biochemistry and Molecular Biology International 39, 21–29. Khalighinasab, M.R., Saify, K., Saadat, M., 2015. Association between GSTM1 and GSTT1 genetic polymorphisms and susceptibility to methamphetamine dependence. Molecular Biology Research Communications 4, 25–32.
n
Corresponding author. Fax: þ98 71 36137432.
Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Letter to the Editor Association between null alleles of GSTM1 and GSTT1 and dependence to heroin and opium To the Editors: Opiates may cause oxidative stress in drug dependent persons (Ghazavi et al., 2013; Soykut et al., 2013). Glutathione S-transferases (GSTs) play an important role in cellular detoxification of xenobiotics. Several GST gene families, including mu and theta, have been identified. The null alleles of genes encoding GSTM1 (a member of class mu; OMIM: 138350) and GSTT1 (a member of class theta; OMIM: 600436) have been well defined in human. In has been reported that GSTT1 and GSTM1 are expressed in the brain (Juronen et al., 1996). Although the association between GSTs polymorphisms and susceptibility to methamphetamine abuse has been reported (Nakatome et al., 2009; Khalighinasab et al., 2015), there are no study investigating the association between these genetic variations and risk of heroin dependence (HD) and opium dependence (OD). Therefore, the present study was carried out. This report consists of two case–control studies performed in Shiraz (southern Iran). In the first study, 442 HD subjects (42 females, 400 males) and 799 healthy controls (137 females, 662 males) were included. A detailed description of these subjects has been reported in our previous report (Saify et al., 2014). In the second study, 143 OD (12 females, 131 males) and 570 healthy controls (55 females, 515 males) were included. All patients were assessed using the Structured Clinical Interview based on Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria for heroine and opium dependence. Moreover, urine drug screens were obtained. All patients were interviewed by a senior psychiatrist. The patients were in methadone maintenance for treating OD and all of them reported opium as their primary drug of choice. Control individuals were blood donors, who declared that they did not suffer from substance abuse. The mean age (S.D.) of the patients and the controls were 39.6 (11.3) and 40.8 (10.7) years, respectively. There was no significant difference in the gender and age distribution between the patient and control groups (P 40.05). The participants were selected from Persian Muslims (Caucasians) living in Shiraz (Fars province, southern Iran). This study was approved by the Shiraz University ethics committee and the informed consent was obtained from each subject. Using the QUANTO (http://biostats.usc.edu/software) software, to detect a real difference in genotypic frequency with a power of 0.80, α ¼ 0.05, OR ¼1.50, and 50% frequency of the GSTM1 null genotype; a minimum sample of 390 would be necessary. With similar assumptions and 25% frequency of the GSTT1 null genotype; a minimum sample of 560 would be necessary. The present case–control studies are more than sufficiently powered to detect a small–medium effect in genotype frequency between cases and their control groups. The PCR conditions for determining the genotypes of GSTT1 and GSTM1 and quality control were previously reported (Khalighinasab et al., 2015). http://dx.doi.org/10.1016/j.psychres.2015.05.004 0165-1781/& 2015 Elsevier Ireland Ltd. All rights reserved.
Table 1 shows that the null genotypes of GSTM1 (OR¼ 0.90, 95% CI: 0.71–1.14, P ¼0.401) and GSTT1 (OR¼ 1.25, 95% CI: 0.95–1.65, P¼ 0.107) were not associated with the risk of HD. After stratification of the participants according to their genders, the same associations were observed. The present study revealed that the GSTM1-null genotype was not associated with the susceptibility to OD (OR¼ 0.69, 95% CI: 0.48–1.00, P ¼0.052). However, the null genotype of GSTT1 was significantly associated with OD (OR ¼1.56, 95% CI: 1.02–2.39, P¼ 0.037) (Table 1). To investigate whether the combinations of the genotypes could alter the risk of substance abuse, we considered the associations between combinations of the genotypes and risks of HD and OD. The reference group consisted of individuals with “null genotype of GSTM1/positive genotype of GSTT1” (Table 1). Statistical analysis revealed that the combination of “positive genotype of GSTM1/null genotype of GSTT1” significantly associated with either HD (OR ¼1.52, 95% CI: 1.03–2.25, P ¼0.034) or OD (OR ¼2.60, 95% CI: 1.44–4.69, P¼ 0.001). Previous studies indicated that heroin and opium dependent persons are under oxidative stress conditions (Ghazavi et al., 2013; Soykut et al., 2013). We know that oxidative stress may occur due to increase in the generation of free radicals or in reduction in the level of antioxidant enzymes. Oxidative stress plays an important role in the pathogenesis of different diseases, especially in the Table 1 Associations between GSTM1 and GSTT1 polymorphisms and dependence to heroin and opium. Genotypes
Controls
Patients
Heroin dependent group GSTM1 polymorphism Positive 367 214 Null 432 228 GSTT1 polymorphism Positive 632 332 Null 167 110 GSTM1/GSTT1 combination genotypes Null/positive 338 176 Positive/positive 294 156 Positive/null 73 58 Null/null 94 52 Opium dependent group GSTM1 polymorphism Positive 255 77 Null 315 66 GSTT1 polymorphism Positive 460 104 Null 110 39 GSTM1/GSTT1 combination genotypes Null/positive 249 50 Positive/positive 211 54 Positive/null 44 23 Null/null 66 16
OR
95% CI
P
1.0 0.90
– 0.71–1.14
– 0.401
1.0 1.25
– 0.95–1.65
– 0.107
1.0 1.01 1.52 1.06
– 0.78–1.33 1.03–2.25 0.72–1.56
– 0.890 0.034 0.758
1.0 0.69
– 0.48–1.00
– 0.052
1.0 1.56
– 1.02–2.39
– 0.037
1.0 1.27 2.60 1.20
– 0.83–1.95 1.44–4.69 0.64–2.25
– 0.265 0.001 0.555
Letter to the Editor / Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
brain. It leads to DNA and cell damage and alter the Ca2 þ channel function (Hool, 2007). Our present findings indicate that the null genotype of GSTT1 (but not the GSTM1 null genotype) is significantly associated with elevation of the risk of heroine and opium dependence. It should be mentioned that at the present time, it is premature to discuss the cause and effect of the GSTM1 and GSTT1 null alleles on the dependency of heroin and opium. Determination of GSTM1 and GSTT1 gene expression levels of cells treated with these drugs are recommended for future experiments. Population stratification and use of blood donors as control group without any verification of whether or not they met criteria for a substance use disorder are limitations of the present study.
Nakatome, M., Miyaji, A., Mochizuki, K., et al., 2009. Association between the GST genetic polymorphisms and methamphetamine abusers in the Japanese population. Legal Medicine 11, 468–470. Saify, K., Saadat, I., Saadat, M., 2014. Association between VNTR polymorphism in promoter region of prodynorphin (PDYN) gene and heroin dependence. Psychiatry Research 219, 690–692. Soykut, B., Eken, A., Erdem, O., et al., 2013. Oxidative stress enzyme status and frequency of micronuclei in heroin addicts in Turkey. Toxicology Mechanisms and Methods 23, 684–688.
Mohammad Rashid Khalighinasab, Khyber Saify, Mostafa Saadat n Department of Biology, College of Sciences, Shiraz University, Shiraz 71454, Iran E-mail address: [email protected] (M. Saadat)
Conflict of interest
Received 18 November 2014 15 April 2015 2 May 2015
No competing interests are declared by any of the authors. Acknowledgments The authors are indebted to the participants for their close cooperation. The authors are indebted to Dr. Raheleh Masoudi for critical reading of the manuscript. This study was supported by Shiraz University (93GCU1M1741). References Ghazavi, A., Mosayebi, G., Solhi, H., et al., 2013. Serum markers of inflammation and oxidative stress in chronic opium (Taryak) smokers. Immunology Letters 153, 22–26. Hool, L.C., 2007. Evidence for the regulation of L-type Ca2 þ channels in the heart by reactive oxygen species – mechanism for mediating pathology. Proceedings of the Australian Physiological Society 38, 87–93. Juronen, E., Tasa, G., Uuskula, M., et al., 1996. Purification, characterization and tissue distribution of human class theta glutathione S-transferase T1-1. Biochemistry and Molecular Biology International 39, 21–29. Khalighinasab, M.R., Saify, K., Saadat, M., 2015. Association between GSTM1 and GSTT1 genetic polymorphisms and susceptibility to methamphetamine dependence. Molecular Biology Research Communications 4, 25–32.
n
Corresponding author. Fax: þ98 71 36137432.
