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.

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.

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.

Mental health status following severe sulfur mustard exposure: a long-term study of Iranian war survivors.

INTRODUCTION: This study aimed to describe the mental health status of sulfur mustard-exposed survivors suffering from severe respiratory and ophthalmological problems. METHODS: Out of 450 invited Iran-Iraq War survivors of sulfur mustard exposure with severe symptoms, 350 participated in this cross-sectional study. Mental health status was assessed using the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, criteria. Fisher exact test, Pearson chi-square test, and chi-square test were used to assess any relationship, and the independent-sample t test was employed to compare differences between the veterans with ocular and pulmonary injuries. RESULTS: There were 60.9% (n = 213) survivors who suffered from mental disorders. Among them, 39.7% (n = 139) were previously untreated and required the initiation of psychiatric treatment. The prevalence of anxiety and mood disorders among all survivors was 40.6% (n = 142) and 32.0% (n = 112), respectively. The most common anxiety and mood disorders were posttraumatic stress disorder (32.9%, n = 115) and major depressive disorder (22.3%, n = 78), respectively. Psychiatric disorders were more prevalent in cases with severe pulmonary chemical injury than in subjects with severe ophthalmologic chemical injury. Significant relationships were found between the types of psychiatric disorders and age, education, and occupation (P < .05). CONCLUSION: The psychiatric morbidity in the chemically injured populations was remarkable and significantly different between the populations. The prevalence of mental illness in these groups highlights the need for the appropriate provision of mental health services.

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 Sulfur Mustard-Exposed Veterans.

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 stabil- ity 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.

Epidemiological Study of Child Casualties of Landmines and Unexploded Ordnances: A National Study from Iran.

BACKGROUND: Despite landmine-risk education programs and extensive demining activities on the Western border of Iran, landmines and unexploded ordnance (UXOs) still cause civilian and child casualties three decades after the Iraq-Iran war (1980-1988). The objective of this study was to understand the epidemiological patterns and risk factors of injury in child casualties of landmines and UXOs in Western and Southwestern Iran. METHODS: Children who were 18 years old or younger at the time of study and who sustained injuries from landmines and UXOs were identified through a search at the Iranian National Veterans Registry. These children participated in a 5-day gathering. The information on socioeconomic status, health-related issues, quality of life, health care utilization, and clinical profiles concerning the landmine and UXO injuries were collected. The method of data collection consisted of three component surveys: health interview, social survey, and medical examinations. Social surveys and health interviews were conducted in a face-to-face method by utilizing a questionnaire consisting of 39 questions addressing household and individual components, including information on time and type of injuries, physical activity, mental health, and quality of life. A comprehensive team of physicians in different subspecialties evaluated and examined children to assess the current medical and psychiatric conditions and physical activity, and recommended and arranged further medical, rehabilitation, or surgical planning. RESULTS: Seventy-eight child casualties were identified and participated in the study. The mean age of the participants at the time of study was 16.11 years old (SD=2 years). The mean age of victims at the time of injury was 8.2 years (SD=3.12 years; ranged from 2 to 15 years old). Sixty-seven (85.9%) of the children were male. Provinces of Kurdistan and Kermanshah had the highest number of casualties, with a total number of 54 children (68.3%). Eighty percent of the injuries were caused by landmines, and UXO explosions were reported in 20% of the cases. Overall, 24 children (30%) had received some landmine-risk education before or after the events. Sixty percent of the explosions had happened in the morning between 9:00 am and 12:00 pm. Playing and grazing livestock were the most prevalent activities/reasons at the time of injury, which were reported in 77% of the subjects. Sixty-three percent of incidents had multiple casualties and in only 13 explosions were the children the only victims of the explosion. The most prevalent injuries were amputations in 41 subjects (52.56%), followed by hearing loss in 23 subjects (29.5%). Amputations were more common in upper extremities (62%) than in lower extremities (38%). CONCLUSION: Landmines and UXOs comprise a significant safety hazard to the children living in the Western border of Iran decades after the Iraq-Iran War. The large number of injuries and lack of risk training among victims suggest that landmine cleanings and landmine-risk education should be age-specifically targeted and expanded substantially.

Quality of life in caregivers of severely disabled war survivors.

PURPOSE: To evaluate quality of life (QOL) of caregivers of severely disabled war survivors and identify variables threatening caregivers' QOL. METHODS: A cross-sectional study was performed on 532 caregivers of Iran-Iraq war related injured survivors by using Persian version of 36-item Short Form Health Survey (SF-36). FINDINGS: Caregivers had lower QOL compared to the Iranian female population (p < .001). Caregivers of amputees had better SF-36 scores compared to caregivers of two groups of chemical warfare survivors (p ranging from .01 to <.001). Caregiving to chemical warfare and being married at the time of trauma were the independent predictors of poor QOL in both the physical component summary (OR = 5.08, 95% CI = 3.35-7.7; OR = 0.57, 95% CI = 0.37-0.89) and the mental component summary (OR = 4.12, 95% CI = 2.68-6.32; OR = 0.63, 95% CI = 0.40-0.98). CONCLUSION AND CLINICAL RELEVANCE: Caregivers of war veterans suffer from poor QOL. Chemical warfare agents contribute to more persistent poor QOL in caregivers than the injuries caused by conventional weapons.

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.

Mustard gas exposure and carcinogenesis of lung.

UNLABELLED: Sulfur mustard (SM), also known as mustard gas, is an alkylating compound used as a chemical weapon in World War I and by Iraqi forces against Iranians and indigenous Iraqi Kurds during the Iran-Iraq War of the 1980s. Although SM is a proven carcinogen there are conflicting views regarding the carcinogenicity of a single exposure. The present study characterizes lung cancers formed in mustard gas victims from the Iran-Iraq War. METHODS AND MATERIALS: Demographic information and tumor specimens were collected from 20 Iranian male lung cancer patients with single high-dose SM exposures during the Iran-Iraq War. Formalin-fixed, paraffin-embedded lung cancers were analyzed by immunohistochemistry for p53 protein. In addition, DNA was extracted from the tissues, PCR amplified and sequenced to identify mutations in the p53 and KRAS genes associated with SM exposure. RESULTS: A relatively early age of lung cancer onset (ranging from 28 to 73 with a mean of 48) in mustard gas victims, particularly those in the non-smoking population (mean age of 40.7), may be an indication of a unique etiology for these cancers. Seven of the 20 patients developed lung cancer before the age of 40. Five of 16 cancers from which DNA sequence data was obtainable provided information on eight p53 mutations (within exons 5-8). These mutations were predominately G to A transitions; a mutation consistent with the DNA lesion caused by SM. Two of the lung cancers had multiple p53 point mutations, similar to results obtained from factory workers chronically exposed to mustard agent. No mutations were detected in the KRAS gene. DISCUSSION: The distinguishing characteristics of lung carcinogenesis in these mustard gas victims suggest that a single exposure may increase the risk of lung cancer development in some individuals.

Amputations due to landmine and unexploded ordinances in post-war Iran.

BACKGROUND: In view of lack of comprehensive data on landmine casualties that lead to amputation in Iran, we conducted this study to determine the pattern and demographic features of landmine explosions that result in amputation of the victims. METHODS: To define the pattern of landmine- and unexploded ordinances-induced amputations and to understand the most common types of underlying activities at the time of the blast, a retrospective study was conducted among the victims in 5 western provinces of Iran, West Azerbaijan, Kermanshah, Kurdistan, Ilam, and Khuzestan between 1988 and 2003. RESULTS: Of a total of 3713 victims, 1499 had undergone amputations. The mean age of the victims at the time of accident was 23 years; 92% of the victims were male, 48.4% of them were of very poor education and all were civilians. Below knee amputation was the commonest type of amputation. CONCLUSION: The occurrence of lower limb amputations from landmine injuries in Iran is a significant burden on the healthcare system; rendering allocation of more resources to provide preventative and rehabilitation measures is therefore a must.

Assessment of fertility among mustard-exposed residents of Sardasht, Iran: a historical cohort study.

BACKGROUND: Mustard gas (HD) is an alkylating agent with mutagenic and carcinogenic effects. Previous reports have demonstrated the ability of this class of compounds to cause adverse reproductive effects, however as of the time of this writing, few correlations have been established between HD exposure and human infertility. In the present study we hypothesize that infertility among mustard-exposed individuals is higher than among the general population. METHODS: 117 couples, 90 with at least one partner, and 27 with both partners exposed to HD in June 1987, were evaluated for occurrence of infertility (defined as the failure to conceive after 12 months of unprotected intercourse). Two groups of subjects were considered in order to establish dose-responsive effect of HD on fertility. One cohort was married at the time of exposure and were evaluated in a time period 12 months from that date. The second cohort married after the date of exposure and each couple was evaluated for a year during a period following marriage. Measurements of these subjects were compared with worldwide incidence of fertility. RESULTS: A 7.5% rate of infertility was observed among couples who were married at the time of exposure; and a rate of 10.3% was noted among individuals single at exposure and subsequently married, for an overall rate of 8.3%, which compares with a worldwide rate of 10-15%. CONCLUSIONS: This study showed that within a population of HD-exposed individuals, elevated environmental levels of the agent during a time period in which couples were actively attempting to conceive, failed to correlate with increased risk of infertility. However, these results must be interpreted with caution based on experimental design which limits the definition of infertility to one 12 month time period.

Incidence of lung, eye, and skin lesions as late complications in 34,000 Iranians with wartime exposure to mustard agent.

Approximately 34,000 Iranians known to have sustained mustard agent exposure during the Iran-Iraq war of 1980-1988 and survived over a decade afterwards were screened for distribution of the most commonly occurring medical problems. In order of greatest incidence, these include lesions of the lungs (42.5%), eyes (39.3%), and skin (24.5%). Within each subpopulation, patients were ranked according to severity of lesions. Twenty-three percent to 37% of patients exhibited at least mild coverage, with 1.5% to 4.5% classed as moderate, and a much smaller population (0.023-1.0%) of the 34,000 patients exhibiting extensive (severe) lesional coverage. These results provide a comprehensive overview of the medical problem most common among mustard victims and could serve as a predictor of the likely impact of these weapons on health status of populations exposed to them during ongoing military conflicts.