Long-Term Methadone Intake and Genotoxicity in Addicted Patients

authors:

avatar Mohsen Rezaei 1 , 2 , * , avatar Forouzan Khodaei 1 , avatar Mehdi Sayah Bargard 3 , avatar Mahsa Abasinia 1

Department of Pharmacology and Toxicology School of Pharmacy, Jundishapur University of Medical Sciences, Ahvaz, IR Iran
Cellular and Molecular Research Center, Jundishapur University of Medical Sciences, Ahvaz, IR Iran
Educational Development Center, Jundishapur University of Medical Sciences, Ahvaz, IR Iran
Jundishapur Journal of Natural Pharmaceutical Products: Vol.10, issue 1; e17008
published online: February 25, 2015
article type: Research Article
received: January 6, 2014
revised: August 10, 2014
accepted: October 6, 2014
DOI: https://doi.org/10.17795/jjnpp-17008

how to cite: Rezaei M, Khodaei F, Sayah Bargard M, Abasinia M. Long-Term Methadone Intake and Genotoxicity in Addicted Patients. Jundishapur J Nat Pharm Prod. 2015;10(1):e17008. https://doi.org/10.17795/jjnpp-17008.

Abstract

Background:

It is well known that contact with some physical, chemical or biological compounds can increase the incidence of mutation. Among these compounds, are pharmaceuticals that meet long duration of use and potentially could be misused and taken more than the ordered dosage.

Objectives:

The aim of this study was to evaluate mutagenic effect of methadone in addicted patients referred to Imam Khomeini Hospital in Ahvaz by single cell gel electrophoresis technic or comet assay.

Patients and Methods:

In this study, 90 subjects were divided into dichromate treated group, no treated healthy volunteers group and test group. Each group included 30 subjects. Screening was performed according to questionnaire and qualified subjects were entered the study. Blood samples were collected and lymphocytes were isolated, mixed with low melting point agarose for slide preparation according to standard method. Slides were analyzed using fluorescence microscope and comet patterns were assessed.

Results:

The mutagenicity index in addicted group was robustly higher than healthy volunteers. Fortunately, this significant difference was lower than positive control.

Conclusions:

Genome instability in addicted patients was demonstrated in this study. Controversially, considering incoherent results of previous studies and our data, more studies in longer duration of methadone use are needed to elucidate the consequence.

2. Objectives

Given controversial results regarding methadone genotoxicity, we investigated the effect of methadone genotoxicity. Altogether, research on mutagenesis of methadone may uncover its potential adverse effects contributing to overall health of patients.

3. Patients and Methods

HEPES, Na2HPO4 and Disodium EDTA were obtained from Sigma (USA), C15H28NO3Na, Tris Hydrochloride and Triton x-100 from Merck (Germany), LMPA and Agarose normal from Fermentas (EU) and Methadone from Exir (Tehran, Iran).

3.1. Patients and Healthy Subjects

This study was performed to evaluate mutagenic effect of methadone in addicted patients referred to Imam Khomeini Hospital in Ahvaz. Total samples of 90 subjects were divided into dichromate treated group, no treated healthy volunteers group and test group. Each group included 30 subjects as follows:

No treated healthy volunteers group: normal subjects without any history of opioid consumption in the last six months. All individuals filled a detailed questionnaire according to the published protocol by the International Commission for Protection against Environmental Mutagens and Carcinogens.

Dichromate treated group: 500 μM of dichromate was added to samples as a positive agent for induction of DNA breakage (13).

Test group: all 30 subjects in this group met the inclusion criteria.

Inclusion criteria were methadone use 60 to 120 mg daily, age of 18 to 65 years, minimum period of three years for methadone consumption and IQ above 75 (10, 11). Consents were obtained from all subjects participated in this study according to university Ethics Committee.

Exclusion criteria were use of any medicines such as benzodiazepines, amphetamine derivatives, antipsychotics, mood stabilizers, use of substances such as marijuana, crack, opiates, tramadol, mental disorders (personality disorders, mood disorders, anxiety disorders, psychoses), organic disease and positive results for HIV Ab , HCV Ab or HBS Ag (7, 10, 11).

3.2. Isolation of Peripheral Blood Lymphocytes

Two milliliters of peripheral blood were collected from each patient in heparinized tubes. Whole blood with Hank's solution was diluted to a ratio of 1 to 2.5 mL of sterile Ficoll solution (in the dark and temperature of 2-8°C), added to a centrifuge tube and diluted blood poured slowly to form a layer on top of the tube. The tubes were centrifuged for 20 minutes 1000 rpm (at this stage centrifugal brake or stopping using hand was avoided). After centrifugation, mononuclear cells (including lymphocytes) settle as a white layer between Ficoll and plasma. Lymphocytes were collected using pipette and transferred to another tube. The tube was filled with Hank's solution and centrifuged at 440 rpm for 10 minutes. The supernatant discarded, deposited again on Hank's solution added and mixed gently and centrifuged again for 15 minutes at 230 rpm. Besides, washing repeated with 110 rpm again. At this point, fully isolated lymphocytes achieved (14).

3.3. Comet Assay

Afterwards, 100 µL of cell suspension premixed with low melting point agarose (LMA 1%) poured on a slide coated with normal melting point agarose (NMA 1%) and covered with cover slips. Slides were kept for 15 to 20 minutes horizontally in the ice tray to solidify. Next, the cover slips were removed from the slides and placed in a lysis solution (14.61 g NaCl, 3.72 g EDTA, 0.125 g Tris ,0.9 g NaOH , 1 g sodium lauryl sarcosinate %1, DMSO 10% , Triton x-100 1% and pH = 10) for one hour and then washed by deionized water, kept for 20 minutes in electrophoresis buffer (NaOH 12 g, EDTA 372.0 g, dH2O to 1000 mL and pH = 13) and electrophoresed at 25 V and 300 mA for 20 minutes and then washed with neutralized buffer (Tris 1.12 g, dH2O to 250 mL and pH = 7.5) for five minutes three times. Slides were immersed in ethidium bromide solution for five minutes and according to the method described by Speit and Hartmann (15), which is based on the original work of Singh et al. (16), slides were analyzed by fluorescence microscope. The extent and distribution of DNA damage indicated by comet assay was evaluated by examining cells. The cells were visually scored into comet classes according to tail size class (17-19): 0 = no tail, 1 = tail shorter than the diameter of the head (nucleus), 2 = tail length 1 to 2x the diameter of the head and score 3 = tail longer than 2x the diameter of the head. Comet without head and those with nearly all the DNA in the tail or with a very wide tail were excluded from the evaluation, because they probably represented dead cells (20, 21). Tail length and the mutagenic index were calculated according the following formula MI = (0 NMC+ 1 SMC+ 2 MMC+ 3 LMC)/200, or we could express it as NMC = No migration cells (score 0), SMC = Short migration cells (score 1) MMC = Medium migration cells (score 2), LMC = Long migration cells (score 3) (22).

3.4. Statistical Analysis

Using SPSS software, Chi-square was performed to compare the groups.

4. Results

Peripheral blood was collected from 30 addicted patients screened to be exclusively on methadone and lymphocytes were separated to assess the genotoxicity by the comet assay. Percent of mutagenicity index (MI) was significantly lower in the test group compared to positive control and conversely, it was higher compared to the negative control group. Addicted patients showed more than two fold MI% compared to negative control or normal samples. Damaged cells in the test group were significantly higher compared to negative control. Altogether, instability of genome was demonstrated for addicted patients according to scores obtained for damaged cells.

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