Sulfur mustard (SM) is a cytotoxic, blistering, and irritant military chemical agent capable of reacting with most biologic molecules of the human body. While it is not a gas, it is a semi- liquid chemical capable to turning into steam. It is chlorosis in its pure form but unstable and carcinogenic when used as a military agent [1]. Upon contact with soil and water, SM evaporates in the air and settles down in minutes to days, depending on the environmental conditions. Since infection signs do not appear immediately and contaminated areas look quite normal, exposed individuals may receive large doses of it [2][3].

Its main acute pathological complications are depigmentation and wet lesions on the skin, lung function disturbances, ophthalmologic manifestations, conjunctivitis, corneal abrasion gastrointestinal symptoms, hematological complications, and bone marrow failures. One of the most catastrophic effects of SM poisoning is suppression of the immune system which leads to lesions and blisters, and results in opportunistic infections [4]-[7]. Slight or moderate exposure is not usually fatal but a long period of treatment is needed; even after remedy for the burns, there is still a great risk of developing cancer. SM and its complications for the human body can be detected with blood and urine tests within a few weeks after the last exposure [2]. It is found in the fat of thigh, abdominal skin, and subcutaneous fat. Lung, spleen, liver, blood, and urine specimens also show low concentrations of unmetabolized SM in patients [8].

Xerosis, or dry skin, refers to sensitive skin which is easily irritated by external stimuli and is, most importantly, itchy. It is often located on the extensor surface of the legs and arms, but in susceptible individuals, it may involve the entire cutaneous surface. Also, it is worse in the winter when humidity is low. Water is lost from the outer most layer of the skin [8]. Dry areas that are repeatedly washed reach a point at which the epidermal barrier can no longer maintain its integrity. Dry skin is an important feature of an atopic state whose characteristics vary with age.

Atopic dermatitis seems to develop from a vicious circle of dermatitis associated with elevated T-lymphocyte activation, hyperstimulatory Langerhans cells, defective cell-mediated immunity, and B-cell mediated IgE overproduction. Triggering factors include temperature change and sweating, decreased humidity, excessive washing, contact with irritating substances, contact allergy, aeroallergens, antimicrobial agents, food, and emotional stress [5][9].

Using Path analysis, the effects of the relationship between exposure and outcome, as impacted by mediators, can be assessed. Path analysis was first used in 1920 for quantitative genetic studies and later in ecology, psychology, social sciences and economics [10][11], as well as health sciences [11]-[13]. It helps to illuminate the relative strength of direct and indirect relationships among a variety of variables. In this statistical approach, a set of causes and effects were diagnosed as a series of steps in a path with a coefficient assigned to each step for quantifying the relationships [14]-[16]. Moreover, Path analysis can be an effective tool to identify the main risk factors of earlier biological effects [17]. Fourteen different methods have been proposed for testing models including intervening variables, and according to them, three general approaches have been offered. These include causal steps in which 4 criteria are tested in the causal step methods [18]-[19]. Due to MacKinnon et al., the independent variable has both direct and indirect effects on the dependent variable (through a mediating variable), if these effects are equal in magnitude but opposite in sign.

The next component is ‘difference in coefficients’, based on comparing two regression or correlation coefficients, for the relationship between independent and dependent variables (skipping mediating variable) and for the relationship between independent and dependent variables after removing the effect of the mediating variable on the dependent variable. Finally, the ‘indirect effect’ of the independent variable on dependent variable, through a mediator, is obtained by multiplying the coefficient for path independent variable-mediator by the coefficient for path mediator-dependent variable [20][21].

Basic and clinical studies have been done on immediate and long-term effects of SM in both animals and human beings [21]-[23], mostly on respiratory and pulmonary systems [24]-[26], skin [27][28], eyes [29], immune system [30][31], urologic and physical conditions of exposed individuals [32][33], and alterations in the serum levels of cytokines, chemokines, immunoglobins, and testosterone [34][35].

Using a Sardasht-Iran historical cohort study (1), the aim of this research was to assess Hemato_4 (HCT), Hemato_5 (PLAT (1000)), Hemato_9 (MCHC), and Biochem_20 (Alkaline Phosphatase; ALK.Phosphat IU/L (IFCC)) as biomarkers and possible mediators of the relationship between Xerosis and SM.

Assuming thatY is Xerosis (present vs. absent), X is study group (exposed to SM vs. non- exposed to SM), and M is mediator (HCT, PLAT (1000), MCHC or ALK. Phosphat IU/L (IFCC) biomarkers) using mediation models, the total, direct and indirect effects of variables using the bootstrap method are indicated in Table 1 , Table 2 , Table 3 and Table 4 . Each shows the accuracy of all effects obtained. The coefficient, significance, and variance are close to that gained from GLMs.

As expected, Xerosis was significantly related to study group (χ2=9.489, p<0.005). HCT was found to be significantly related to study group (R=0.132, F (1,466) =8.287, p<0.0042 (using linear regression, β=1.046, p<0.0042)). Xerosis was significantly related to HCT (using logistic regression, Exp (β) =1.093, p=0.011). Xerosis was significantly related to the combination of HCT and study group (using logistic regression, Exp (β) =1.082, p=0.026, Exp (β) =2.187, p=0.01 respectively). Direct and indirect effects of Exposure on Xerosis explained by HCT are shown in Table 1 .

PLAT (1000) was found to be significantly related to study group (R=0.22, F (1,466) =23.767, p=0.000) using linear regression (β=-27.857, p=0.000). Xerosis was significantly related to PLAT (1000) (using logistic regression Exp (β) = 0.995, p=0.036). Xerosis is not significantly related to the combination of PLAT (1000) and study group (using logistic regression; Exp (β) =0.996, p=0.119, Exp (β) =0.013, p=0.013, respectively). Direct and indirect effects of Exposure on Xerosis explained by PLAT (1000) are shown in Table 2 .

MCHC was found to be significantly related to study group (R=0.107, F (1,466) = 5.371, p=0.021; using linear regression, β=-0.316, p=0.021). Xerosis was significantly related to MCHC (using logistic regression, Exp (β) =0.817, p=0.019). Xerosis was significantly related to combination of MCHC and study group (using logistic regression, Exp (β) =0.833, p=0.037, Exp (β) =2.236, p=0.008, respectively). Direct and indirect effects of Exposure on Xerosis are explained by MCHC, as shown in Table 3 .

ALK.Phosphat IU/L (IFCC) was not found to be significantly related to the study group (R=0.09, F (1,467) = 3.82, p=0.051; using linear regression, β=-15.96, p=0.051). Xerosis was not significantly related to ALK.Phosphat IU/L (IFCC) (using logistic regression Exp (β) = 0.996, p=0.08). Xerosis was not significantly related to combination of ALK.Phosphat IU/L (IFCC) and study group (using logistic regression Exp (β) =0.997, p=0.176, Exp (β) =2.267, p=0.007, respectively). Direct and indirect effects of Exposure on Xerosis, as explained by ALK.Phosphat IU/L (IFCC), are shown in Table 4 .

It is more rational to have a set of biomarkers as a potential set of mediators or multiple mediators. Using bootstrap of 1000 at α= 0.05 showed significant indirect effects of SM on Xerosis mediated through two biomarkers (HCT and MCHC), with increased false positive error. Indeed, 90.46% of the effect of SM on Xerosis is direct without being mediated through HCT. This effect could have been mediated through other biomarkers not included in the model. In the pathway of SM on Xerosis, some other important mediating variables should be considered and assessed as better mediators- PLAT (1000), MCHC, and ALK.Phosphat IU/L (IFCC).

SM may not have any direct effect on Xerosis, but it can impose indirect effects through HCT and other biomarkers. Indeed, if these biomarkers are not included in the model, then the indirect effect of SM on Xerosis may be mistaken as a direct effect of SM on Xerosis [24].

The bootstrap method showed valid computations, in addition to the 9.54%, 11.6%, 6.69%, and 5.32% of the mustard sulfur (MS effects) on Xerosis, as explained by HCT, PLAT (1000), MCHC, and ALK-Phosphat IU/L (IFCC), respectively. The partial mediating effects of HCT and MCHC are also included. Indeed, all variables considering in a multiple mediating model could result in better mediation validity since SM affects several organisms at the same time.

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SM: Sulfur Mustard

The authors declare no conflict of interest.

Department of Biostatistics, Tarbiat Modares University, Tehran, Iran.

This study, based on (part of a cohort data), is a cohort study, available on the website of the Iranian Ministry of Health. Notice the reference number one that points to this issue.

Will be provided if request.

All authors contributed to the design of the research. MZ, SF, SJ and MS collected the Data. ZKH, MZ and SF conducted analysis and interpretation of data. All authors drafted the first version. MZ, SF, SJ, and MS edited the first draft. All authors reviewed, Commented and approved the final draft.