Our study found that the overall frequency of physical abnormalities is significantly associated with children whose fathers were exposed to mustard gas. Furthermore, there was a significant association between paternal exposure to mustard gas and both respiratory diseases and congenital malformations. Given the widespread use of mustard gas in wartime, the lasting effects may potentially last generations.
Animal and human studies indicate that paternal exposure to certain agents can result in developmental abnormalities in progenies [16]. Several studies demonstrated that exposure of male rats and mice to cyclophosphamide can lead to congenital malformations in progenies [17, 18]. In humans, paternal exposure to various agents such as acrylamide, lead, and solvents result in an increase in congenital malformations [19–21]. A mechanism suggested for the effect of paternal preconception exposure is the occurrence of transmissible genetic changes or an epigenetic mechanism [22–24].
Possible explanation of findings
Sulfur mustard is a cytotoxic agent with mutagenic and carcinogenic effects [15]. Its active intermediate, sulfonium ion, reacts rapidly with proteins and nucleic acids, alters chemical functional groups such as amines, carboxyls, phosphates, S-H, and O-H groups, and produces alkylation products. This process may result in cross-linking between adjacent strands of DNA, which has been shown to be extremely lethal to cells [6];
Confirmed effects of sulfur mustard gas on spermatogenesis may explain the observed overall increase in physical abnormalities among the progenies of chemical victims [2, 12, 25, 26]. However, few studies, previously performed, were conclusive in determining a causal relationship. Pour-Jafari et al. [27] studied the rate of congenital malformations among progenies and their parents of Iranian victims before and after chemical warfare exposure and found that the rate of major malformations has increased from 33 per 1000 to 258 per 1000. Although they had used his cases as their own controls, and thus reduced selection bias, they did not adjust for the effect of parents' increasing age on malformation occurrence. Taher et al[28] claimed that the use of mustard gas in the Iran-Iraq conflict might have increased the number of cleft lip and cleft palate in children, however, they were not able to establish any causal effect between these two events, nor could they exclude the effects of other possible causes.
Strengths & Limitations
Strengths of our study include its sample size and locally relevant controls. Sardasht is one of the rare instances in the world with a large population of mustard gas victims. Despite initial resistance to studying the effects of exposure, overtime, initial politico-ethical resistance has faded. No similar study has yet been performed with this population. Furthermore, presence of an unexposed population in a nearby city (Rabat) with characteristics similar to the exposed group provided a unique opportunity to further strengthen this analysis. An inherent limitation of studies like this, where the participants may consider probable benefits by over-reporting adverse outcomes, and also where a long time is passed from the date of the event under investigation, is the possibility of recall bias. We aimed to minimize these biases by an inclusive physical exam and appropriate paraclinical studies performed by GPs and further confirmation by a pediatrician. However, our exposed group included only the progenies of those exposed males who were present in the city at the time of study. Moreover, we do not know the number of men exposed that died or moved since exposure. We used clinical examination and paraclinical tests of live birth children to determine disorders and malformations. It is possible that kariotyping and other genetic studies could have revealed more problems [25, 29]. It is also possible that miscarriages or abortions would have yielded differing effects. Finally, it is possible that our control population differs importantly from the exposed population that we have not recognized. This issue exists with any non-randomized comparison and we are unable to overcome this concern.
Our study found a significant association between exposure to mustard gas and common disorders and malformations. We did not find that any specific disorder or malformation was associated with exposure. We expected this as, with anencephaly, for example, there is a prevalence rate of about one in 1000 live births [1], thus, even with a doubling of risk rates, a much greater population would need to be studied to reveal strong association with chemical exposure. Considering the high number of chemical victims in Iran, this study may catalyze further comprehensive assessments with larger study populations. Using a post hoc sample size calculation, we find that our study had greater than 95% power to detect malformations and greater than 99% power to detect malformations/disorders.
We evaluated all clinical disorders and malformations, regardless of hypothesis driven associations with chemical exposure. Our reasoning for this is that the link between genetic disorders and resulting illnesses is not yet completely understood. So, for example, while rheumatic fever with congenital involvement may be most often associated with communicable disease genesis, we cannot rule out that parental chemical exposure may be associated with predispositions to certain illnesses [30].
Interpretation
Our study found a significant association between overall frequency of physical abnormalities and disorders and paternal exposure to mustard gas. Given the considerable victims of mustard gas among Iranian civilians and military personnel, as well as civilians and military in other conflicts, the effects of war may have a lasting and important effect on generations to come.
Ethical statement
The Board of Research Ethics in the Janbazan Medical and Engineering Research Center (JMERC) and Shahed University approved this study. Informed written consent was obtained from all the parents and caregivers involved in this study.
