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Effect of met-enkephalin on chromosomal aberrations in the lymphocytes of the peripheral blood of patients with multiple sclerosis.

INTRODUCTION

Met-enkephalin is one of the simplest endogenous opioid peptides within the enkephaline family. Endogenious opioid peptides share amino sequence of tyrosine-glycineglycine-phenylalanine (aka Opioid motif), and contain one or more copies of met-enkephalin (Tyr-Gly-Gly-Phe-Met) and leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Opioid receptors (OP) are detected in human phagocytic leukocytes, with a direct binding of naloxone in lymphocytes and thrombocytes [1]. Met-enkephalin binds with high affinity to OP1 (5) receptors, and with low affinity to OP3 ([mu]) receptors. Additionally, it specifically binds to receptors on T lymphocytes which are not morphin receptors [1, 2]. As a potential receptor on human lymphocytes a complementary transcript of met-enkephalin is isolated, with a single sequence that matches cytokine receptor y chain [3]. Multiple sclerosis (MS) is a progressive disease followed by development of the neurological deterioration. Relapsing/ remitting form of the disease is highly sensible to immunosuppressive therapy. However, with the extended duration, the response-rate to the treatment tends to decrease as well. As a result of an assumption which claims the existence of the inflammation and of the neurodegenerative phase, patients with MS are recommended for early immunomodulatory treatment [4, 5]. Due to immunomodulatory properties met-enkephalin was applied in clinical studies and it effect on the stabilization of the clinical conditions of MS was documented [6, 7]. It also manifests in vivo citoprotective effects [8]. Met-enkephalin mostly induces immunostimulation when applied in low doses, and immunosuppresion when applied in higher doses. Higher doses of met-enkephalin exerted suppressive effect in experimental treatment of allergic encephalomyelitis [2, 9]. A role of the released cytokines and of Thi cells differentiation disorder are implied in an immuno mediated demyelization [4]. The research of the cerebrospinal fluid in MS patients reveals an increase in the levels of immunoglobulin (lg) and mononuclear pleocytosis. Furthermore the same studies showed the numerous somatic gene mutations in the variable region of the lg Heavy Chain in the cerebrospinal fluid B cells in MS patients [10,11]. Research by Stambuk et al. [3] detected a significant reduction in the frequencies of the structural chromosome aberration in human lymphocytes of the peripheral blood of MS patients.

MATERIALS AND METHODS

The research was conducted at the Neurological Clinic of the Clinical and University Center of Sarajevo, and the Center for Human Genetics of the Faculty of Medicine, University of Sarajevo.

Samples

Blood samples were obtained from seven female patients in relapse in a test tube containing heparin. The eligibility criteria were MS diagnosis as per McDonald Diagnostic Criteria, depicted on existence of objective proofs of at least two lesions (MRI or evoked potentials), or at least two clinically diagnosed symptomatic disease episodes. Patients included in the study were never treated with interferon, and had not received pulse corticosteroid treatment over the past six months.

Tested substance

Met-enkephalin (Biotechnology Laboratories Richmond, USA) was dissolved in distilled water and kept at a temperature of -18[degrees]C. Prior to adding to culture, it was kept at a room temperature (18-23[degrees]C) up to five minutes. The met-enkephalin concentration per culture 2 (C2) was 1.2 [micro]g/ml and per culture 3 (C3) 120 [micro]g/ml. Control culture (C1) was not incubated with the tested substances.

Research design

Blood samples were cultivated following method described by Moorhead et al. [12], with the incubation of cultures during 72 hours and the application of Colcemid stock solution 25 mcg/ml (0.2 ml) two hours before completion of incubation period. After microscopic analysis of chromosome preparations by standard procedure (Giemsa staining), the identification of rearranged chromosomes was conducted by destaining and applying the G-band technique. The total number of chromosomes included in structural aberrations was determined in the following manner: numerical analysis did not include chromosomes with gaps; its number was separately analyzed. It is deemed that the single chromosome was included when the following structural aberration existed: chromosome/chromatide break, acentric fragment, ring fragment, minute, acentric ring, ring chromosome, marker chromosome. Two chromosomes are deemed included when the following structural aberration existed: dicentric chromosome and translocation.

Statistical analysis

The collected data was statistically processed by computer software SPSS v.11 (Statistical Package for Social Sciences[C], March 2004). For the purpose of the hypothesis testing, we used a non-parametric testing for correlated samples, Wilcoxon Signed Ranks Test. Findings from control culture (C1) and cultures incubated with various concentrations of tested substances (C2, C3) were compared.

RESULTS

Female patients included were 34 to 60 years old (41.89 [+ or -] 9.17), while the total number of hospitalizations due to MS was from one up to 6 hospitalizations (2.67 [+ or -] 1.80). The recorded values of fibrinogen ranged between 9.10-15.90 [micro]mol/L (12.44 [+ or -] 2.52).

Our research reviewed 200 mitosis per each tested culture. The total number of chromosomes included in structural aberrations are presented in Table 1. and detected aberrations in Figure 1.

Among detected structural aberrations the highest presence of gaps, breaks and marker chromosomes was documented, The ring chromosomes and the chromosome fragmentation were present within the C1 only; while dicentric chromosomes were detected in C1 and C2. When control cultures were compared to incubated ones, no statistically significant differences were recorded either in the number of chromosomes included in structural aberrations (C1 vs C2, p=0.527; C1 vs C3: p=0.089) or in the number of mitosis with aberrations (C1 vs C2, p=1.000; C1 vs C3, p=0.581). The identified chromosomes included in structural chromosome aberrations are displayed in Appendix 1. The frequency of engagement of certain chromosomes in aberrations, expressed in percentages, is shown in Table 2. For the calculation of frequency of associated aberrations with a familiar origin, the following were included: gap, break, translocation, chromosome fragmentation, dicentric and ring chromosomes. The majority of translocated marker chromosomes was detected in C1, while chromosome 14 was mostly included in translocations. Basic descriptive statistics for the numerical aberrations is displayed in Table 3. The number of all detected numerical aberrations is listed in Figure 2. When control cultures were compared to incubated ones, a statistically significant increase in number of numerical aberrations was detected in the incubated cultures (C1 vs C2, p=0.027; C1 vs C3, p=0.039). When observing polyploidy, the most fequent was the presence of endoreduplication, while triploidy and tetraploidy were somewhat rarer. Additionally, hyperdiploidy was detected. Furthermore, after G-banding the most frequent engagement in polysomy was of the X chromosome. Among the detected aneuploidy, the trisomy and tetrasomy of the X chromosome were dominant (Figure 3). When control cultures were compared to incubated ones, a statistically significant increase in the number of poliploidy was detected in the culture treated with a lower concentration of met-enkephalin (C1 vs C2, p=0.034), while no significant difference was documented when controls were compared to cultures treated with higher concentration of met-enkephalin (C1 vs C3, p=0.131). No statistically significant difference existed in the number of endoreduplications (C1 vs C2, p=0.157; Ci vs C3, p=0.334). A statistically significant increase in the number of aneuplody existed in cultures incubated with lower met-enkephalin concentration compared to control cultures (Ci vs C2, p=0.026). No statistically significant difference was revealed when a culture incubated with a higher concentration was compared to control ones (C1 vs C3, p=0.236). Mitotic index was determined as a percent of lymphocytes in mitosis (M1+M2), counted on 300 lymphocytes. No statistically significant difference in mitotic index existed between the control culture and the cultures incubated with various concentrates of substance used for testing (C1 vs C2, p=0.674; C1 vs C3, p=0.753).

DISCUSSION

Our research detected disappearance of ring chromosomes and chromosome fragmentations in the cultures treated with met-enkephalin. Similar to our results, the study by Stambuk et al. [3] showed disappearance of ring chromosomes and chromosome fragmentation after in vitro treatment of cultures with met-enkephalin (1.2 [micro]g/mL) and incubation period of 48 hours. However, in contrast to their results, our research did not reveal significant effect of met-enkephalin on the reduction of the number of structural aberrations and on disappearance of dicentrical chromosomes. Furthermore, within the five-days cell culturing with incorporated 3H-thymidine, Stambuk et al. [3] documented significant reduction in the number of cells reaching the third stage of mitosis and a significant increase in a number of first metaphase. Chromosomal fragile sites expressed through an increased frequency in gaps and breaks are identified, as well as a presence of conservation of fragile sites throughout evolution [13, 14]. Re et al. [14] suggested that fragile sites via modulated gene expression can participate in the regulation of the cell responsivity rate to oncogenic stress and DNA damage. Ilyinskikh et al. [15] calculated the expected frequency of chromatic aberrations which are induced by radiation (8.44 up to 2.04 from the first up to 22nd chromosome), while the number of breaks increases with an increase in absolute chromosome length. The longest chromosomes in our research were also most frequently included in aberrations (chromosome 1 in C1 and C2, and chromosome 3 in C3). The frequency of chromosome 9 in structural aberrations was the most prominent. It is difficult to interprete the noticed impact of the met-enkephalin on the number of aberrations in treated cultures. The relationship between the ploidy disorders in malignant cells and an increase in the cell growth potential was suggested [16]. A hypothesis on aneuploidy as chromatic base of cancerogenesis was established [17]. Our research detected dominant engagement of X chromosome in hyperploidy and polysomy. According to data obtained from the Mittleman Data Base on chromatic aberrations in malignant diseases [18], the trisomy of X chromosome is most frequently related to acute lymphoblastic leukemia and lymphoblastic lymphoma. Previous research using met-enkephalin do not point out cancerogenous potential of this peptide; rather it shows quite the opposite [19-22]. When applied with paclitaxel, met-enkephalin enhances the inhibition of tumor growth of squamous cells head and neck carcinoma [19, 20]. Furthermore, throughout the experiment, aneugenic potential of opioids morphine and noskapine was detected [21, 22]. Genotoxic in vitro effects of noskapine were not confirmed in vivo, probably due to fast metabolism and low systemic bioavailability of the medication [21]. Experimental study by Cheng et al. [23] also suggests that met-enkephalin inhibits cell proliferation of various human and animal cells--probably by inducing an expression of inhibitors of cyclin-dependant kinases.

CONCLUSION

In conclusion, although the application of met-enkephalin in the culture of peripheral blood lymphocytes of MS patients did not manifest statistically significant protective effects, it influenced the disappearing of serious structural aberrations such as ring chromosomes and fragmentation of chromosomes. Met-enkephalin showed an impact on the number of numerical aberrations in both treated cultures, which certainly demands further in vivo evaluation.

DECLARATION OF INTEREST

The authors declare no conflict of interest.

APPENDIX 1. Identified chromosomes for cultures not
treated with met-enkephalin

       P1            P2          P3          P4

1      1r (p;q),                 Gap 1q      Break 1q
         Break 1p,
         Gap1q
         and 1q
2      Gap 2q        t(2q;14q)               t(2p;14q),
                                               Gap 2q
3      Gap 3q                                Gap 3q
4                                            Gap 4q
5                    Break 5p    Gap 5q
6      Gap 6q                                Break 6p
7      Gap 7q
8      Gap 8q        Frag 8,
                       Gap 8q
9      Break 9q                              Gap 9q
         and 9q,                               i 9q
         Gap 9q
         and 9q
10                   Gap 10q
11     Break 11p                             Dic(Xq;11q),
                                               Gap 11p

12                   Frag 12

13

14                   t(2q;14q)               t(2p;14q)
18                   Gap 18q

X                                            Dic(Xq;11q)

       P5            P6          P7

1      Gap 1q,                   Gap 1q
         1q i
         1q

2

3                    Break 3p    Gap 3q
4
5                    Break 5p
6
7
8                    Gap 8q      Break 8q

9                                Break 9q,
                                   Gap 9q

10
11

12                   Dic(12q;
                       18q)
13     Break                     Break 13q
         13q
14                               Break 14q
18                   Dic(12q;
                       18q)
X                                X(q24)

Index: t-translocation, p-upper row, q-lower row,
c-centromere area, frag-fragmentation Identified
chromosomes in cultures treated with met-enkephalin

       P1            P2          P3          P4

Culture 2

1      Dic(1p;12q)   Gap 1q,     Break 1q    t(1q;2q)
         Break 1q      1q
                       and 1q

2      Gap 2q        Gap 2p                  t(1q;2q),
3      Break 3q                                Mar 2p
         and 3q
4
5                                            Break 5q
7      t(7q;14q)                 Gap 7q
         Break 7p
8
9      Gap 9q                    Break 9p    Break 9q

10                               Gap 10p
11                                           Gap 11q
12     Dic(1p;12q)               Break         and 11q
                                   12q and
                                   12q,
                                   Gap 12p
14     t(7q;14q)
17     Break 17q                             Gap 17q
X

Culture 3

1                                Break 1p    Gap 1(c)
                                               and 1q
2      Break 2p                  Break 2q    Break 2p
2(c)   Break 2q      Gap 2q
3                                            Break 3q

4      Break 4q
5      Break 5p                  Break 5p    Break 5q
6                    Gap 6q
7      Gap 7q                                Gap 7p
8      Break 8q
         and
         Gap 8p
9      Gap 9q                    Gap 9q,
         and 9q                    9q
                                   and 9q
10                                           Gap 10q
11                                           Break 11q
13     Gap 13q
19     Break 19p
20
X      Break Xp

       P5            P6         P7

Culture 2

1      Gap 1q                   Break 1q
                                  and 1q,
                                  Gap 1q
                                  and 1(c)
2      Gap 2(c)      Gap 2(c)   Break 2p
3                    Gap 3(c)

4      Break 4q
5                               Break 5q
7      t(7q36;14)

8                               Break 8p
9      Break 9p,     Gap 9q     Break 9q
         Gap 9q        and 9q     and 9q,
                                  Gap 9q
                                  and 9q
10                              Gap 10q
11
12     Gap 12q                  Break 12p

14     t(7;14q12)
17
X      Break Xq,                Break Xp
         Gap Xp

Culture 3

1                               Gap 1q

2      Gap 2p and
2(c)
3      t(3p;4q),
         Break 3p,
         Gap 3p
4      t(3p;4q)
5
6
7
8      Gap 8q                   Break 8p
                                  and 8q,
                                  Gap 8p
9      Break 9q,
         Gap 9q

10
11
13     Gap 13q
19                   Gap 19q
20                              Mar 20
X                               Gap Xq

Index: t-translocation, p-upper row, q-lower row, c-centromere
area, frag-fragmentation


ACKNOWLEDGEMENTS

The authors want to acknowledge Farmacija d.o.o., Tuzla for providing us with an opportunity to work in this area by donating the tested substances as well as to Ms. Amra Catovic for technical and other support during the research process.

REFERENCES

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Maida Rakanovic-Todic (1) *, Lejla Burnazovic-Ristic (1), Slavka Ibrulj (2), Nedzad Mulabegovic (1)

(1) Institute of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Sarajevo, Cekalusa 90, 71 000 Sarajevo, Bosnia and Herzegovina. (2) Center for Cytogenetics and Molecular Medicine, Faculty of Medicine, University of Sarajevo, Cekalusa 90, 71 000 Sarajevo, Bosnia and Herzegovina.

* Corresponding author: Maida Rakanovic-Todic Institute of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Sarajevo, Cekalusa 90, 71 000 Sarajevo, Bosnia and Herzegovina Phone/Fax: +387 33 227 018

e-mail: maida@dic.unsa.ba

Submitted: 26 March 2013/Accepted: 21 February 2014

TABLE 1. Descriptive statistics for structural chromosomes
aberrations

Culture   N   [bar.X] [+ or -] SD   Median   Xmin   Xmax

C1        7   6.14 [+ or -] 2.67      5       4      12
C2        7   5.29 [+ or -] 4.68      4       0      13
C3        7   4.29 [+ or -] 1.98      4       1      7

TABLE 2. The frequency of associated chromosomes in
structural aberrations, expressed in percentages

Chromosome      C1(%)   C2(%)   C3(%)

1               17.24   19.35   8.00
2               6.90    11.29   14.00
3               6.90    4.84    8.00
4               1.72    1.61    3.00
5               5.17    3.23    6.00
6               3.45     --     2.00
7               1.72    6.45    3.00
8               8.62    1.61    12.00
9               13.79   17.74   14.00
10              1.72    3.23    2.00
11              5.17    3.23    2.00
12              3.45    9.68     --
13              3.45     --     3.00
14              5.17    3.23     --
15               --      --      --
16               --      --      --
17               --     3.23     --
18              3.45     --      --
19               --      --     3.00
20               --      --     2.00
21               --      --      --
22               --      --      --
X               3.45    4.84    3.00
Unidentified     n=5     n=4     n=5
  chromosomes

Aberration not detected in a certain chromosome

TABLE 3. Descriptive statistics for numerical chromosome
aberrations

Culture   N        [bar.X]         Median   Xmin   Xmax
Code             [+ or -] SD

C1        7   3.29 [+ or -] 2.69     2       1      9
C2        7   7.00 [+ or -] 2.83     7       3      12
C3        7   5.14 [+ or -] 4.91     4       2      16
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Author:Rakanovic-Todic, Maida; Burnazovic-Ristic, Lejla; Ibrulj, Slavka; Mulabegovic, Nedzad
Publication:Bosnian Journal of Basic Medical Sciences
Article Type:Report
Geographic Code:4EXBO
Date:May 1, 2014
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