A review on delayed toxic effects of sulfur mustard in Iranian veterans

Iranian soldiers were attacked with chemical bombs, rockets and artillery shells 387 times during the 8-years war by Iraq (1980–1988). More than 1,000 tons of sulfur mustard gas was used in the battlefields by the Iraqis against Iranian people. A high rate of morbidities occurred as the result of these attacks. This study aimed to evaluate the delayed toxic effects of sulfur mustard gas on Iranian victims. During a systematic search, a total of 193 (109 more relevant to the main aim) articles on sulfur mustard gas were reviewed using known international and national databases. No special evaluation was conducted on the quality of the articles and their publication in accredited journals was considered sufficient. High rate of morbidities as the result of chemical attacks by sulfur mustard among Iranian people occurred. Iranian researchers found a numerous late complications among the victims which we be listed as wide range of respiratory, ocular, dermatological, psychological, hematological, immunological, gastrointestinal and endocrine complications, all influenced the quality of life of exposed victims. The mortality rate due to this agent was 3%. Although, mortality rate induced by sulfur mustard among Iranian people was low, variety and chronicity of toxic effects and complications of this chemical agent were dramatic.

Mustard gas or sulfur mustard is a chemical warfare agent (King of the battle gases) [1] with cytotoxic, vesicant and blistering effects on exposed skin [2]. This agent can enter the body through various routes including the skin, the respiratory system, conjunctiva and the gastrointestinal system by contaminated foods [3]. It can cause both acute and delayed manifestations and late complications even 40 years after the exposure as reported for victims of the first worldwar [4]. People can be exposed to small or large amounts of mustard gas through terroristic actions, wars, leakage from the factories, and even activities like fishing [5].
Sulfur mustard can be easily produced. It has a delayed, extensive absorption, multi-organ effects and quick penetration. It is stable in the environment, has a low production cost, is easy to use and has the ability to debilitate soldiers [6,7]. After dissemination in an area, mustard gas remains in that area for a long time and makes that area non-habitable. Mustard gas victims temporarily lose their vision a few hours after exposure which is a critical strategy in warfare [7], and so far, it has been used in >10 military conflicts [8].
Human losses related to destructive effects from this agent in the World War l are as follow: the soldiers were only equipped with protective breathing masks and lack of skin protection resulted in the death of more than 90,000 soldiers and about 1.3 million injured people [8] and out of which, 400,000 people required long term medical care [9]. According to the other reports mustard gas caused 14,000 injuries in the first 3 months of use in World War I and a total of 120,000 injuries [10]. In the US army, out of the total of 36,965 chemical warfare victims, 27,711 (75%) were due to sulfur mustard gas, and according to a report from the contamination control unit of the British army, of a total of 160,970 chemically injured soldiers, 124,752 (77.5%) had been injured by sulfur mustard gas. The greatest damage by chemical warfare agents in World War I was done to the Russians resulting in 50,000 casualties and 500,000 injuries. Years after the application of mustard gas in World War I, its delayed progressive and destructive effects were recognized [7].
In the beginning of Iraq-Iran war in 1980, use of chemical warfare agents was limited but in March 1985, despite international conventions on prohibition of using chemical warfare agents, Iraqis extensively used these agents against Iran. They used nerve agents (Sarin & Tabun) and mustard gas against Iranian soldiers [11,12].
According to data reported by Veterans and Martyrs Affair Foundation (VMAF) which is responsible for taking care of the war victims in Iran, the Iranian people were 387 times attacked with chemical bombs, rockets and artillery shells during the 8-years war by Iraq (1980)(1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988) [13]. More than 1,000 tons of sulfur mustard was used in the battlefields [2] and about 100,000 of people were injured due to this agent [8], as at the present, after more than two decades, still about 30,000 of them are under treatment [14].
Sardasht is a city in north-west of Iran. Iraqis released four 250 kg bombs containing mustard gas on this city at 4 pm, July 27, 1987 injuring 4,500 civilians [15]. Use of mustard gas against Iranian soldiers by the Iraqis was reported to the international commissions by Iran in 1984 and in 1986 application of this agent by Iraqis was documented by the United Nations observatory team led by M. Dominguez [16]. They evaluated the battlefields, clinically examined the victims and performed some para-clinical tests and approved that Iraqis had used chemical bombs containing mustard gas and to a lesser extent organophosphorous nerve agents especially Tabun [17].
Mustard gas was used by the Iraqis in south and west of Iran many times and in 1988 the last chemical attack was performed on Oshnaviyyeh city injuring 2,680 civilians [13][14][15][16][17][18].

Definition and etiology
When mustard plants with the Latin name, Synapsis is affected by the tyrosine glycoside Synacrine enzyme, a substance is produced that naturally does not exist in the seeds of this plant. This compound is isothiosyanate which it is a vesicant agent [19]. Mustard compounds are classified among the alkylating agents that substitute hydrogen with an alkyl cation. A large group of these compounds such as nitrogen mustards (chlorambucil) is mainly used in medicine for treatment of cancers. The other group namely sulfur mustard is considered a chemical warfare agent used in many battlefields in the 20 th century [3]. This agent is recognized with various abbreviations H, HD, HT, LOST, SM (sulfur mustard), MG (mustard Gas) and Y perit "H" and "HD" come from the words "Hun Stuffe" and "Distilled", HT is a combination of 6% HD and 40% of a substance called T and the LOST come from the name of the scientists Lommel and Stein kopf, who developed a method for the large-scale production of the agent for the German army. French say to this agent, Y perit because it was used in the Y pres at the first time [20].

Pathology and mechanisms of actions
In summary, this alkylating substance leads to DNA damage, cell membrane damage, decrease in glutathione, activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), and caspase activation. DNA damage lead to Poly (ADP-ribose) polymerase (PARP) activation and nicotinic adenine nucleotide (NAD) depletion and this process in turn will lead to necrosis. Glutathione decreasing produce reactive oxygen and these two phenomena may lead to necrosis and cell death [27]. In addition to cell death, SM has many other 1860 Frederick Guthrie noted its blistering properties [1,8] 1886 Victor Meyer produced pure sulfur mustard. [1] 1917-1918 First use in World War I by the German army -along 10 days about 1 million sulfur mustard shells were poured on Belgian soldiers. 1.3-2.5 million people in Belgium and France and thousands of British soldiers were injured. [1,8,10] 1919 Production of mustard gas in American factories reached 19 tons a day. [7] 1925 Use of these agents was banned in Geneva Gas Protocol. [8] 1935-1936 Italy breached the Geneva Protocol treaty and used sulfur mustard gas against Ethiopia when conquering the Ethiopian plane. [8]

1943
A cargo ship carrying a large amount of sulfur mustard exploded in the harbor of Bari, Italy. The gas was disseminated in the area injuring more than 600 people. [1] 1937-1945 Japan used sulfur mustard gas against China. [8,10] 1945-1948 A large amount of sulfur mustard was poured in the Baltic Sea. Exposure of Scandinavian fishermen to this agent resulted in development of skin blisters [1] 1963-1967 Egypt used sulfur mustard bombs against Yemeni pro-monarchy supporters. [1,8] 1983-1988 Iraq extensively used SM and nerve agents against Iran and injured more than 50,000 victims. [8,9] 1988 Iraqi army killed 5,000 Iraqi civilians in Halabcheh using SM. In this attack, nerve agents like sarin were also used. [1,8] adverse effects on cells such as alkylation effects, mitosis inhibition (effects on hematologic system, immunologic system, epithelial and germinal tissues), mutagenesis, carcinogenesis, and colinomimethic effects [28]. On the basis of Kehe et al. study, in summary, mustard gas in the molecular level induces the releasing of cytokines, prostaglandines, matrix metalloproteinases (MMPs) and serine proteases, and increases DNA damages, oxidative stress, and impaired energy metabolism. Following to these molecular changes, cellular infiltration, apoptosis and necrosis occur that is continued by erythema and pain and formation of vesicles, blisters, ulcer and impaired wound healing [27].

Methods
During a systematic search, a total of 193 medical articles related to SM were reviewed using known international medical databases such as Scopus, Medline, ISI, and Iranian medical databases such as Iranmedex, SID, and Irandoc. One hundred and nine articles were more relevant to the main aim. Eight articles were on general aspects of SM effects, 36 articles were related to respiratory effects, 16 articles were on dermatologic effects, 15 articles were on ophthalmologic effects, 11 articles on psychological effects, 10 articles on endocrinology & reproductive health effects, 4 articles related to quality of life and 9 articles were related to other items such as:  plastic breathing masks and rubber or plastic clothing can protect the body for a few hours) and remains active for a long time in cold or moderate temperatures.

Absorption in the body
Eighty percent of the SM gas is evaporated and the remaining 20% penetrates the body.
Of the 20%, 12% remains on the skin and 8% is absorbed systemically. Absorption is done through moist tissues like respiratory system, axillary area, genital area/groins and eyes. Tissues with higher metabolism are more sensitive to this toxic gas. [1,3,8,26] Metabolites The main metabolite of SM in the urine is thiodiglycol which can be detected by chromatography with1ng /ml sensitivity. [3] Excretion Fifty percent of the absorbed SM in the body is conjugated with aminoacid lecithin producing di-cystylethyl sulfone which is excreted through the kidneys. [22]

Anti-toxin
Unfortunately there is no specific antitoxin for SM gas [6] Applications Except for chemical warfare, a nitrogen analogue of SM is now being used in chemotherapy for treatment of leukemia. This therapeutic agent is called Mustargen [1] neurologic, oncologic, hematologic, cardiologic, laboratory. No special evaluation was conducted on the quality of the reviewed manuscripts and the credit of journal was considered sufficient. Our study had been handled in accordance with the rules of the ethical review board of Tehran University of medical sciences.

Initial description of victims
Some Iranian soldiers described their first encounter with SM as follows: immediately after exposure to the SM bomb, we smelled a garlic odor and had a bitter taste in our mouth. A few hours later, we became dizzy, with a headache and we could not breathe. A short time after the explosion, we developed small bleeding areas and inflamed small lesions (macules and papules) on our skin. Then we felt hoarseness in our voice and this condition has been going on and off so far [28,29]. Also, some other victims described the events at the exposure time as follows: We were exposed to SM gas delivered through bomb explosion from 5 to 30 meters distance. We smelled a strong smell of garlic, addled egg or fried vegetables. A bluish-grayish cloud and a white dust appeared in the sky. We had no protection. In the first hours following the attacks, we washed our faces and hands with waters which we did not know the water may be contaminated with SM [14]. Gradually, ocular, dermatologic and respiratory symptoms developed. We are still suffering from the related respiratory symptoms.
Mustard effects on body organs are divided into acute and chronic phases. Most of the reviewed articles were performed in chronic phase of exposure which they are summarized in Table 3.
In a study conducted on 100 Iranian chemical warfare victims, 94% had dermatologic, 94% had ophthalmic, 75% had pulmonary, 5% had GI and 10% had hematologic complications as the result of chemical exposure [16].
In another study, delayed toxic effects of mustard gas were evaluated in 236 Iranian chemical victims 2 to 28 months after exposure and complications were as follows: respiratory complications in 78%, CNS in 45% and dermatologic complications in 24.5% of cases [35]. Table 3 shows distribution of pulmonary, ophthalmic and dermatologic complications in several studies in Iran [10,15,[30][31][32][33][34].
According to report of Khateri and colleagues, the pulmonary, ophthalmic and dermatologic complications were the most common in 34,000 victims observed [30].

Complications
Mustard gas complications in Iranian victims were as follows:

Other complications include
Decreased quality of life and sleep disorders as the result of several physical (ophthalmologic injuries, pruritus, COPD and . . .) and mental complications [9,54], contamination of the wives of exposed soldiers [3,55]. Mustard gas also can cause liver and kidney failure, skin cancer, and bone marrow depression [56]. McNamara and colleagues in their study on rats found that using 0.5 to 2 mg/kg mustard gas through the stomach tube in days 6 and 15 of pregnancy caused no evidence of teratogenicity [15,57]. The most prevalent GI complications in Iranian victims were nausea, vomiting, loss of appetite, stomach pain and diarrhea. Mustard gas can cause stomach cancer, basal cell carcinoma, Bowen's carcinoma and spinocellular carcinoma [1]. Mustard gas can eventually cause death. However, the mortality rate is low (2 -3%) [8,58].

Discussion
According to initial description of the victims, there is a latent phase between the exposure and appearance of symptoms. This time interval increases the morbidity of exposed victims. Victims at first are not aware of their exposure/contamination and as the result they do not attempt to clean themselves which consequently results in greater absorption of the toxic agent into the tissues. This phase usually takes between 30 minutes to 8 hours [8].
To deal with this phase soldiers should train on how to deal with chemical terrorism, especially with mustard gas. For example, they should avoid washing their hand, face and body with water in the area that was attacked because it may be contaminated with the poison.
Longitudinal studies showed that those exposed to mustard gas suffer from long term complications causing significant morbidity [8]. There are 2 types of complications: early and late. Early complications appear in the first week following exposure but late complications may manifest in the next 50 years following exposure [10]. In chronic phase, patients may be suffering from various diseases.
During World War I, scientists found that mustard gas can cause bone marrow aplasia, dissolution of lymphoid tissue and GI ulcers. Later many more complications were reported due to mustard gas including respiratory, dermatologic, ocular, GI, hematopoietic, endocrine, neural, psychological, ear-nose-throat (ENT), genital, reproductive and immune system [21] involvement which can manifest as early or delayed complications.
Mustard gas is a chemical warfare agent that is detrimental for the earth and has a significant toxic effect on microorganisms in the soil. It also inhibits the enzymatic activity of the soil [65]. On the other hand, SM can remain in the battlefields (for example beside the moats in World War I) and can be found in the amount of 1-25 mg/m3 in 6-12 inch depth of the soil. In moderate temperatures with mild winds SM can remain stable for more than a week [8]. Different forms of SM can be stored in the soil for up to 10 years [10]. Considering all the above facts, since the war zones in our country now are major tourist areas, and every year a large number of people travel to these areas, healthy soil-building, should strictly be placed on the agenda of the popular visits.
There are different reports about the prevalence of death and its causes in Iranian veterans. Ghanei et al., in their study have stated that, the rate of death among 1,005 people who died during their study period, were 72% by diseases, 16.1% due to unintentional accidents, 2.3% due to suicide, and 0.8% caused by intentional accidents. The causes of deaths in 2.8% were uncertain and 12.1% of deaths were related to chemical agents effects [66].
Tavallai et al., in their study conducted on 1239 Iranian veterans also reported that 65.4% of deaths were due to diseases, and 24.7% were due to accidents [67].
In summary, reported fatality rates in exposed soldiers in World War 1 were less than 2% and among Iranian victims were 3-4% and disabling potential of SM is much greater than lethality of this agent [68]. According to Tavallai report, the most common causes, had led to death were cardiac diseases and this according to an other report, in Iranian victims 75% of deaths were due to respiratory complications [18], and many of Iranian soldiers lost their lives in a chemical battle scene and probably the cause of death was pulmonary edema induced by chemical agents (29).

Conclusion
Although, mortality rate induced by sulfur mustard among Iranian people was low, the variety and chronicity of adverse effects and complications of this chemical agent were dramatic.