@2024 Afarand., IRAN

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ISSN: 2008-2630    Iranian Journal of War & Public Health 2015;7(1):1-6

Background
Mustard gas analogues are aerosols which remain in the victims’ body and cause different disabilities via affecting skin, eyes, and respiratory system [1, 2]. … [3] Early complications of exposure to mustard gas are revealed in the first week, while its late complications are revealed 10-15 years later or even more years after the first complication [4, 5]. … [6-12] As a hemostatic mechanism to save cell population in the tissues and as a defense mechanism in the reactions of the immune system, or when the cells are damaged by toxic agents and diseases, apoptosis, naturally, occurs during development and aging [13].
Previous Studies
Mustard gas causes sensitivity to mutation in tumor suppressors (such as p53), which decrease bcl2 and activates caspase-3 in vitro [14]. Mustard gas induces apoptosis death in NHBE and SAEC in vitro cell culture [15]. In lung tissues of rats exposed to mustard gas, expression of the genes related to apoptosis has increased [14]. … [16-20]
Aim(s)
The aim of this study was to compare the apoptosis rate in blood mononuclear cells of chemical victims 25 years after exposure to mustard gas with non-exposed persons.
Research type
This is a case-control study.
Research society, place and time
Chemically-injured veterans exposed to mustard gas and non-exposed persons, including healthy persons according to medical examinations and with no specific disease or a documented history of exposure to mustard sulfur, were studied in 2014.
Sampling method and number
10 veterans were selected as “case group” by random sampling method. 11 non-exposed persons were selected as “control group” using random sampling method. All samples were male, aged between 35 and 62 years. Exclusion criteria were person’s unwillingness to participate and loss of samples due to little measure or inappropriate content.
Used devices&materials
From each person, 3ml of peripheral blood were collected in both groups. Mononuclear cells of peripheral blood were separated using Gradient Ficoll method. 10µl of the cells were counted. 200 thousands cells were poured in cell-culture sinks. To the desired ratios, control and target cells were added by the desired ratio and were finally reached to 200µl. 3.5-4h incubation was done at 37°C and 5%CO_2. After removing the supernatant liquid via centrifugal procedure, 200µl cell lysis buffer were added to the remaining cell sediments on the bottoms of the previous sinks and 30min incubation was done at 15-25°C. Apoptosis was measured using cell death detection ELISA kit (Roche; Germany). At first, 20µl of the supernatant liquid, cell lysis solution, and positive control (from the kit) were poured on the center of coated micro-plates of the kit. 80µl of immunoassay reagents (incubation buffer, anti-histone- biotin, and anti-DNA-peroxidase) were added to every sink. After covering with the labels inside the kit, the micro-plates were placed on a shaker (300rpm) for 2 hours and at 15-25°C. Then, the sinks were completely emptied and they were washed 3 times by 250-300µl incubation buffer. 100µl of ABTS solution (from the kit) were added to every sink and it was placed on the micro-plate shaker (250rpm) for 10-20min (upon color formation). Then, 100µl of ABTS stopping solution were added to every sink. Absorption of the sinks at 405nm was compared with “100µl of ABTS stopping solution+100µl of ABTS solution” (as control) [21]. Data were analyzed using SPSS 18 software and T-student Statistical Test.
Findings by Text
There was no significant statistical difference between the mean values of apoptosis rate in control group (0.533±0.168) and case group (0.345±0.116).
Main comparison to the similar studies
… [22] Mustard gas induces apoptosis in human lung epithelial cells and caspases 3 pathway and 8 is highly activated. Inhibition of caspase 3 severely reduces activities of caspases 8 and 9. Inhibition of caspase 8 reduces activity of caspase 3 and completely inhibits activity of caspase 9 [15]. Mustard gas induces apoptosis via Fas respond and siRNA inhibits activity of induced caspase 3 by mustard gas. Both apoptosis and necrosis types of death participate in cell death induced by mustard gas [23]. All the above cases have been focused on acute phase of exposure to mustard gas, while the present study was focused on cases 25 years after exposure to mustard gas. DNA damage due to mustard gas (25 years after exposure) has been reported [24]. Fas/FasL signaling pathway, which leads to apoptosis, has been damaged in BAL cells of the patients exposed to mustard gas [14]. Despite decrease in the number of BCL2 protein, its expression has increased in macrophages of lung biopsy specimens of veterans exposed to mustard gas. In addition, the number of lymphocytes has increased, while there is no change in BCL2 expression as compared to non-exposed persons. In addition, in neutrophils, neither the number of BCL2 protein nor its expression has changed. However, in epithelial cells and fibroblasts, its expression has increased [25]. The above studies have shown the effects of mustard gas on the cells and functional mechanisms of different molecules in the lung cells as a specific tissue. However, the present study was done on mononuclear peripheral blood cells and no significant difference was observed.
Suggestions
More studies of apoptosis in tissues damaged by mustard gas should be done via flow cytometry and immunohistochemistry methods.
Limitations
The veterans hardly agreed to participate in the study.
Conclusions
Chronic phase of mustard gas (25 years after exposure) does not affect the apoptosis rate of mononuclear cells of peripheral blood.
Acknowledgments
The researchers feel grateful to Immunoregulation Research Center.
Conflict of interest
Non-declared
Ethical Permissions
As a part of SCIS project, the study was approved by Ethic Committee of Janbazan Medical and Engineering Research Center.
Funding Sources
The study was funded by Immunoregulation Research Center.