Deoxyribonucleic acid damage in Iranian veterans 25 years after wartime exposure to sulfur mustard

• Background: More than 100,000 Iranian veterans and civilians still suffer from various long-term complications due to their exposure to sulfur mustard (SM) during the Iran–Iraq war in 1983–88. The aim of the study was to investigate DNA damage of SM in veterans who were exposed to SM, 23–27 years prior to this study. • Materials and Methods: Blood samples were obtained from the veterans and healthy volunteers as negative controls. Lymphocytes were isolated from blood samples and DNA breaks were measured using single-cell microgel electrophoresis technique under alkaline conditions (comet assay). Single cells were analyzed with “Tri Tek Comet Score version 1.5” software and DNA break was measured based on the percentage of tail DNA alone, or in the presence of H2O2 (25 μM) as a positive control. • Results: A total of 25 SM exposed male veterans and 25 male healthy volunteers with similar ages (44.66 ± 6.2 and 42.12 ± 5.75 years, respectively) were studied. Percentage of the lymphocyte DNA damage was significantly (p < 0.01) higher in the SM-exposed individuals than in the controls (6.47 ± 0.52 and 1.31 ± 0.35, respectively). Percentages of DNA damage in the different age groups of 35–39, 40–44, 45–49, and 50–54 years in SM-exposed veterans (5.48 ± 0.17, 6.7 3 ± 1.58, 6.42 ± 0.22, and 7.27 ± 0.38, respectively) were all significantly (p < 0.05) higher than the controls (1.18 ± 0.25, 1.53 ± 0.22, 1.27 ± 0.20, and 1.42 ± 0.10, respectively). The lymphocytes incubated with H2O2 had much higher DNA damage as expected. The average of tail DNA is 42.12 ± 2.75% for control cells + H2O2 and 18.48 ± 2.14% for patients cells + H2O2; P < 0.001. • Conclusion: SM exposure of the veterans revealed DNA damage as judged by the comet assay

DNA damage and repair proteins in cellular response to sulfur mustard in Iranian veterans more than two decades after exposure

Delayed effects of sulfur mustard (SM) exposure on the levels of five important damage/repair proteins were investigated in 40 SM-exposed veterans of Iran-Iraq war and 35 unexposed controls. A major DNA damage biomarker protein – phosphorylated H2AX – along with four DNA repair proteins in cell response to the genome damage MRE11, NBS1, RAD51, and XPA were evaluated in blood lymphocytes from the veterans and controls using western blotting. Mean levels of XPA, MRE11, RAD51 and NBS1 were lower in SM-exposed patients and the decrease in NBS1 was significant. Even though the raised level of phosphor-H2AX in SM-poisoned group compared to the controls was not significant it was consistent with DNA damage findings confirming the severity of damage to the DNA after exposure to SM. There were correlations between the values of RAD51 and NBS1 proteins as well as XPA and MRE11 proteins. More than two decades after exposure to SM, there is still evidences of DNA damage as well as impaired repair mechanisms in cells of exposed individuals. Such disorders in cellular level may contribute to long term health problems of the SM veterans

Telomere shortening associated with increased levels of oxidative stress in sulfur mustard-exposed Iranian veterans

Sulfur Mustard (SM) is the most widely used chemical weapon. It was used in World War 1 and in the more recent Iran-Iraq conflict. Genetic toxicity and DNA alkylation effects of SM in molecular and animal experiments are well documented. In this study, lymphocytic telomere lengths and serum levels of isoprostane F2α were measured using q-PCR and enzyme immunoassay-based methods in 40 Iranian veterans who had been exposed to SM between 1983-88 and 40 non-exposed healthy volunteers. The relative telomere length in SM-exposed individuals was found to be significantly shorter than the non-exposed individuals. In addition, the level of 8-isoprostane F2α was significantly higher in the SM-exposed group compared to controls. Oxidative stress can be caused by defective antioxidant responses following gene mutations or altered activities of antioxidant enzymes. Chronic respiratory diseases and infections may also increaseoxidative stress. The novel finding of this study was a the identification of ‘premature ageing phenotype’. More specifically, telomere shortening which occurs naturally with aging is accelerated in SM-exposed individuals. Oxidative stress, mutations in DNA repair genes and epimutaions may be among the major mechanisms of telomere attrition. These findings may help for a novel therapeutic strategy by telomere elongation or for validation of an exposure biomarker for SM toxicity

Mental health disorders in child and adolescent survivors of post-war landmine explosions.

BACKGROUND: To describe the mental health status of 78 child and adolescent survivors of post-war landmine explosions. METHODS: Child and adolescent survivors of landmine explosions who were younger than 18 years old at the time of the study were identified and enrolled in this study. The mental health status of the participants was assessed by general health assessment and psychiatric examinations. Psychiatric assessment and diagnosis were undertaken using the Diagnostic and Statistical Manual for mental disorders (DSM-IV) criteria. A psychiatrist visited and interviewed each survivor and identified psychiatric disorders. RESULTS: Seventy-eight child and adolescent survivors with a mean age of 16.11 ± 2 years old were identified and agreed to participate in the study. The mean age of the victims at the time of injury was 8.2 ± 3.12 years old (range 2-15). Thirty-seven (47.4 %) of the adolescent survivors suffered from at least one psychiatric disorder. Twenty-nine survivors (37.1 %) were newly diagnosed and needed to start medication and psychiatric treatment. The most common findings were anxiety disorders (34.6 %), including posttraumatic stress disorder (PTSD) in 20 (25.6 %), and generalized anxiety disorder (GAD) in 7 (9 %) subjects. Mild-Moderate depression was found in 5 (6.4 %) subjects. No personality disorders were observed, and two patients suffered from mental retardation. The study results revealed a significant association between age of casualty, duration of injury and limb amputation, and types of psychological disorders. CONCLUSION: Child and adolescent survivors of landmine explosions had a high prevalence of psychiatric disorders

Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of

Objective: In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims. Materials and Methods: In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene. Results: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. Conclusion: We demonstrate that non-miRNA reference genes have the least stability in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers

MicroRNA expression in serum samples of sulfur mustard veterans as a diagnostic gateway to improve care

Sulfur mustard is a vesicant chemical warfare agent, which has been used during Iraq-Iran-war. Many veterans and civilians still suffer from long-term complications of sulfur mustard exposure, especially in their lung. Although the lung lesions of these patients are similar to Chronic Obstructive Pulmonary Disease (COPD), there are some differences due to different etiology and clinical care. Less is known on the molecular mechanism of sulfur mustard patients and specific treatment options. microRNAs are master regulators of many biological pathways and proofed to be stable surrogate markers in body fluids. Based on that microRNA expression for serum samples of sulfur mustard patients were examined, to establish specific microRNA patterns as a basis for diagnostic use and insight into affected molecular pathways. Patients were categorized based on their long-term complications into three groups and microRNA serum levels were measured. The differentially regulated microRNAs and their corresponding gene targets were identified. Cell cycle arrest, ageing and TGF-beta signaling pathways showed up to be the most deregulated pathways. The candidate microRNA miR-143-3p could be validated on all individual patients. In a ROC analysis miR-143-3p turned out to be a suitable diagnostic biomarker in the mild and severe categories of patients. Further microRNAs which might own a link to the biology of the sulfur mustard patients are miR-365a-3p, miR-200a-3p, miR-663a. miR-148a-3p, which showed up only in a validation study, might be linked to the airway complications of the sulfur mustard patients. All the other candidate microRNAs do not directly link to COPD phenotype or lung complications. In summary the microRNA screening study characterizes several molecular differences in-between the clinical categories of the sulfur mustard exposure groups and established some useful microRNA biomarkers. qPCR raw data is available via the Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110797

MicroRNA expression in serum samples of sulfur mustard veterans as a diagnostic gateway to improve care

<div><p>Sulfur mustard is a vesicant chemical warfare agent, which has been used during Iraq-Iran-war. Many veterans and civilians still suffer from long-term complications of sulfur mustard exposure, especially in their lung. Although the lung lesions of these patients are similar to Chronic Obstructive Pulmonary Disease (COPD), there are some differences due to different etiology and clinical care. Less is known on the molecular mechanism of sulfur mustard patients and specific treatment options. microRNAs are master regulators of many biological pathways and proofed to be stable surrogate markers in body fluids. Based on that microRNA expression for serum samples of sulfur mustard patients were examined, to establish specific microRNA patterns as a basis for diagnostic use and insight into affected molecular pathways. Patients were categorized based on their long-term complications into three groups and microRNA serum levels were measured. The differentially regulated microRNAs and their corresponding gene targets were identified. Cell cycle arrest, ageing and TGF-beta signaling pathways showed up to be the most deregulated pathways. The candidate microRNA miR-143-3p could be validated on all individual patients. In a ROC analysis miR-143-3p turned out to be a suitable diagnostic biomarker in the mild and severe categories of patients. Further microRNAs which might own a link to the biology of the sulfur mustard patients are miR-365a-3p, miR-200a-3p, miR-663a. miR-148a-3p, which showed up only in a validation study, might be linked to the airway complications of the sulfur mustard patients. All the other candidate microRNAs do not directly link to COPD phenotype or lung complications. In summary the microRNA screening study characterizes several molecular differences in-between the clinical categories of the sulfur mustard exposure groups and established some useful microRNA biomarkers. qPCR raw data is available via the Gene Expression Omnibus <a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110797" target="_blank">https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110797</a>.</p></div

Graphical abstract.

<p>Graphical abstract.</p

The 9 top affected cellular pathways influenced from the 29 microRNAs of the normal-severe comparison.

<p>The 9 top affected cellular pathways influenced from the 29 microRNAs of the normal-severe comparison.</p