**2. Subjects and methods**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

PAHs are known environmental pollutants with harmful effect on human health. Benzo [*a*] pyrene (B[a]P) is a lead compound in this group and one of the most studied carcinogenic PAHs [8]. B[a]P is formed during incomplete combustion of organic materials and pyrolysis of inorganic compounds [9] B[a]P is also found in cigarette smoke [10], cooked food [11], and various combustion gases such as vehicle exhaust [12]. It is also generated from some industrial operations such as those of cooking ovens, heavy oil plants [13] and asphalt plants [14]. Given its harmful effects on health, B[a]P was classified by the International Agency for Research on Cancer (2012) among the highly genotoxic compounds and categorized to "group 1 carcinogenic to humans" [15].

Asphalt workers are exposed to B[a]P through two major ways: though inhalation of emanating fumes from the chimney for those working in the mixing plants, or through inhalation of the vaporous gas while paving asphalt at roadside workshops [16]. Notably, when asphalt is being prepared for road paving, the mixture reaches very high temperature ranging between 130° and 145°, which results emission of large of amount of vaporous fumes, that workers inadvertently inhale. To a lesser extent, some workers are exposed to B[a]P via touching B[a]P-containing compounds [16]. This particularly occurs among mechanics that are often exposed to petroleum compounds. According to the occupational safety and health administration, the allowed concentration of B[a]P in environment when workers perform their duty should not exceed 0.2 mg/ and exposure should not exceed more than 8 hours a day [17]. Although B[a]P is metabolized in all human tissues, the hepatic catabolism of B[a]P through cytochrome P450 (specifically cytochrome P450 1A1) results in production of several metabolites such as epoxides, dihydrodiols, phenols and quinones. First, B[a]P undergo phase I metabolism into epoxides. Subsequently, these epoxides undergo further metabolism though one of three ways: through spontaneous rearrangement to phenols, through hydration to transdihydrodiols by epoxide hydrolase or though phase II detoxification. Phase II dextoxification occurs via binding to glutathione, which can occur spontaneously or though catabolism by glutathione-S-transferases [18]. Given this extensive hepatic metabolism, the amount of B[a]P that reaches the systemic circulation is minimal. Therefore, gastro-

intestinal route appears to be a less important route of exposure to B[a]P.

450 1A1 to form DNA-adducts, that play key roles in its carcinogenesis [9].

B[a]P appears a carcinogenic compound which is metabolized by cytochrome

The mechanism of carcinogenesis of B[a]P is dependent on a 3-step enzymatic metabolism discussed above (**Figure 1**) [18]. These steps eventually form the final mutagen BPDE [19], which is a very reactive and binds covalently to lipids, protein and DNA to form BPDE adducts [20, 21]. These BPDE-DNA adducts can result in permanent mutations if not promptly repaired, which can lead to development of cancer. In this study, we examine the relationship between being an asphalt worker and detection of BPDE-DNA adducts in the white blood cells (WBC), which can be a

**78**

**Figure 1.**

useful surrogate exposure risk.

*B[a]P converts to the final carcinogen, BPDE.*

This was a cross-sectional study that aimed to evaluate the exposure to asphalt fumes (i.e., by measure the hemostatic blood CBC, and Aspartate Amino Transeferase AST), and to evaluate the exposure to B[a]P through asphalt fumes among asphalt workers by determining the levels of BPDE-DNA adducts in their WBC. It was conducted on 25 male workers exposed to asphalt fumes and 25 healthy male individuals without such exposure.

### **2.1 Comparison groups**

**Group 1 (study group):** This group consisted of 25 male individuals who worked at an asphalt plant in Syria. Among those, 12 subjects worked in the mixing plant (9 were employed to monitor the equipment operation and 3 mechanics to maintain the equipment). The remainder 13 subjects were paving workers at roadside workshops (5 subjects to operate the equipment, 4 mechanics and 4 manual construction workers). These subjects were exposed to B[a]P vapors and aerosols of bitumen for many years ranging from 3 to 31 years. On average, all subjects worked 8 hours a day, 4 days a week. Their mean age was 46 years (range: 33–58 years).

The subjects within the study group were carefully selected to avoid external bias. All subjects did not drink alcohol. They all had somewhat comparable dietary habits, that consisted predominantly of vegetables. All subjects did not work outside their primary site of employment. Subjects with significant non-occupational exposure to PAHs (e.g. use burning woods for heat) or those who work a second job were excluded.

**Group 2 (control group):** The control group consisted of 25 healthy volunteers. These individuals were predominantly university administrators and hospital workers. They did not work outside their primary site of employment. There all had no significant occupational or personal exposure to PAHs (e.g. did not use burning woods for heat). They all took no prescription medications and lived remotely from bitumen fumes. The subjects in the control group were selected to match the subjects in exposure group in gender, age, smoking status and dietary habits.

**Subgroups:** Subjects in the study group were categorized into subgroups based on age, and smoking status. Based on age, they were categorized by age: ˂45 years (8 workers, ranging from 33–44 years) and ≥ 45 years (17 workers, ranging from 46–58 years). Based on smoking status, they were categorized to smokers (22 subjects) and non-smokers (3 subjects).

**Study protocol:** All subjects signed written consent prior to enrollment in the study. The study protocol was approved by the Ethical Committee of Damascus University prior to the start of the study.

The study was begun with a questionnaire, that was administered to all subjects to collect general personal information such as age, weight, health status, smoking status, dietary and lifestyle habits and other demographic data.

After enrollment of qualified subjects, blood samples were collected via cubital venipuncture and 5 mL of blood were collected from each subject and placed in sterile tubes. For each subject, one of these tubes was placed immediately in ice bags at 4° and transported immediately to the hospital to be stored in the freezer at −80° until the staff were ready to analyze it. The remainder of the specimen was used immediately to perform various laboratory biomarkers such as complete blood count CBC; WBC, red blood cells RBC, hematocrit (HCT), hemoglobin (HGB) and mean corpuscular hemoglobin concentration (MCHC) using (Sysmex Cooperation, Japan) and (AST) (reference range 0–35 unit/L) using (Biosystem S.A. Spain).

All blood samples were collected in April, 2016.
