Detection of Benzo[a] pyrenediol epoxide-DNA adducts in White Blood Cells of Asphalt Plant Workers in Syria

 

Razan Zohairee¹, Mohammad Amer Zamrik², Sophie Barguil³

¹Master Student DPT of Pharmacology and Toxicology, Faculty of Pharmacy, Damascus University.

²Professor DPT of Pharmacology and Toxicology, Faculty of Pharmacy, Damascus University.

³Assistant Professor DPT of Pharmacology and Toxicology, Faculty of Pharmacy, Damascus University.

 

ABSTRACT:

Background: Benzo [a] pyrene is considered a classic DNA-damaging carcinogen and is a lead compound of polycyclic aromatic hydrocarbons.B [a] Pis metabolically activated by cytochrome P450 enzymes to formation electrophilic metabolites such as epoxides. These epoxides might bind to DNA and produce DNA-adducts. ELISA method is used for determination of DNA adducts of benzo [a] pyrenediolepoxide (BPDE). Objectives: The aim of this study was to evaluate exposure to B [a] P in asphalt plant workers by measuring the BPDE-DNA adducts in their peripheral white blood cells (WBC), which are considered biological markers for exposure risk assessment. MaterialandMethods: In this study, the levels of BPDE-DNA adducts were measured in DNA samples of WBC obtained from asphalt plant workers in Syria and compared to those measured from a control group. The measurement was performed using BPDE-DNA Adducts ELISA kit. The sample size was determined to be 50 with 25 asphalt plant workers and 25 healthy volunteers with no occupational exposure to Benzo [a] pyrene. Results: BPDE-DNA adducts were detected in WBC of 11 asphalt plant workers with concentrations ranging between 0 and 2.75 ng/ml and only one individual in the control group with concentration of 0.75 ng/ml. These results indicate a significant positive relationship betweenexposure toB [a] P through the bitumen fumes (working in asphalt plant) and formation of BPDE-DNA adducts. Conclusions: BPDE-DNA adducts area potential biomarker for PAHs exposure and likely helpful indicator of PAH-induced DNA damage.

 

 

 

INTRODUCTION:

Polycyclic aromatic hydrocarbons (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[1]. B[a]P isformed during incomplete combustion of organic materials and pyrolysis of inorganic compounds [2], B[a] Pis also found in cigarette smoke[3]. cooked food, [4] and various combustion gases such as vehicle exhaust[5].

 

 

It is also generated from some industrial operations such as those of cooking ovens, heavy oil plants ^[6], and asphalt plants^[7]. 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 "group1carcinogenic to humans"^[8].

 

Asphalt workers are exposed to B[a]P through two major ways: through 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[9]. 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.

 

B[a] P is a pro-carcinogen compound that is metabolically activated by cytochrome P450 enzymes, cytochrome P4501A1 (CYP1A1) has been considered to play a central role in the activation step, which is essential for the formation of DNA adducts.

 

The mechanism of carcinogenesis of B[a]Pis dependent on a 3-step enzymatic metabolism (Fig.1) to the final mutagen benzo[a]pyrenediol epoxide (BPDE)^[10]. Very reactive, BPDE binds covalently to proteins, lipids, and DNA to produce BPDE adducts^[11][12]. If left unrepaired, DNA adducts may lead to permanent mutations.

 

 

Figure1 B [a] P converts to the final carcinogen BPDE.

 

The aim of this study was to evaluate exposure to B[a]P in asphalt plant workers by measuring the BPDE-DNA adducts in their peripheral white blood cells (WBC), which are considered biological markers for exposure risk assessment.

 

MATERIAL AND METHODS:

This research was a cross-sectional study that aimed to evaluate the exposure of B[a] P among asphalt workers by evaluating the levels of BPDE-DNA adducts in their WBC. It was conducted on 25 male workers exposed to bitumen fumes and25 healthy male individuals without such exposure.

 

Study Groups:

MonitoringGroup:

This group consisted of 25 male individuals who worked at an asphalt plant in Syria. These subjects were exposed to B[a] P through bitumen fumes 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). Smoking status was restricted to current smoking and non-smoking (3 non-smokers and 22 smokers).

 

The Control Group:

Consisted of 25 healthy volunteers non–occupational exposure to B[a]P and bitumen fumes, they matched to workers, according to gender, age, food habits and smoking (10 non-smokers and 15 smokers).

 

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.

 

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 tubesin 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.

All blood samples were collected in April, 2016.

 

DNA Isolation:

Genomic DNA was isolated from peripheral WBC by using DNA Kit (Thermo Scientific Gene JET Genomic DNA Purification Kit) (Qiagen, USA). DNA concentrations were measured using Gene Nano Drop (Biochrom, England), which occurred in the biology laboratory at Alassad University Hospital in Damascus, Syria. Repeated thawing and freezing of the samples were avoided.

 

Determination of BPDE-DNA adduct Levels:

After DNA was isolated from WBC, the samples were subsequently diluted to a concentration of 2 𝜇g of DNA in 1 ml.Phosphate-buffered Saline with PH 7.2 (1X) (Gibco by life technologies) was used for dilution and washing. BPDE-DNA adduct levels (ng/ml) were measured according to the standard method provided by OxiSelect BPDE-DNA Adduct ELISA Kit (Cell Biolabs, Inc., San Diego, USA). This ELISA kit is an immunoassay enzyme developed for rapid detection of BPDE-DNA adducts.The quantity of BPDE adduct in DNA samples is determined by relative comparison of a known BPDE-DNA standard curve. The kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown DNA samples.The results were expressed as nanograms of BPDE-DNA adducts per microgram of DNA. The analyses were applied twice.

 

Apparatus: ELISA (Tecan, Switzerland) in the Blood Bank of Damascus University.

 

Statistical Analysis:

The statistical analysis of this study was performed using SPSS software version 13.0.The P value of<0.05 was considered to be statistically significant.

 

RESULTS:

BPDE-DNA adducts Levels in the Study Group:-1

In the Monitoring Group, BPDE-DNA adducts were detected in the WBC in 11 out of 25 individuals (44%),

2 non-smokers and 9 smokers. The concentrations of BPDE-DNA adducts among the exposed group ranges between 0 to 2.75 ng/ml except for one individual who had exceptionally high concentration of 16.5 ng/ml . He was a smoker.

 

In the Control Group,

BPDE-DNA adducts were detected in only one individual with concentration of 0.75 ng/ml (Table 1). He was a smoker.

 

Using statistical analysis, the number of individuals with detected BPDE-DNA adducts in their WBC was higher among theMonitoring Group compared to the Control Group (χ2 test, 44 vs 4%, p=0.001).

 

Table1: Levels of BPDE-DNA adducts

 

2-BPDE-DNA adduct levels, according to age:

Among asphalt workers, the concentrations of BPDE-DNA adducts range from 0 to 2.75 ng/ml for subjects<45 year-old and ranges from 0-2.25 ng/ml for those≥45 year-old except for one subject with unusually high concentrations of 16.5 ng/ml. He was from the older age category. Statistically, there was no significant difference in the adducts concentrations between the younger and older age groups (χ2 test, p=0.18).

 

3-BPDE-DNA adducts Levels ,according to Smoking Status:

In the monitoring group, there was no significant difference in the number of individuals with detected BPDE-DNA adducts between smokers and non-smokers (Mann-Whitney U test, P=0.43).

 

DISCUSSION:

In this study, the results showed correlation between BPDE–DNA adduct levels and B[a]P exposure through bitumen fumes of the asphalt workers by inhalation (P=0.001), which poses BPDE-DNA adducts as a potential surrogate marker for prior exposure to B[a]P.These results were compatible with, (Pavanello et al,1999). Pavanello demonstrated significant relationship between chronic inhalation of high levels of PAHs and detection of BPDE-DNA adducts.[13]

 

On the other hand, age did not significantly correlate with the presence of BPDE-DNA adducts among subjects in the study group (p=0.18).These results are in agreement of those reported by(McClean et al, 2006)[14] and is likely due to the similar metabolic and excretion ability in both age groups.

 

Interestingly, although smoking has somewhat similar effect on DNA as exposure to B[a]P, this study showed no correlation between smoking and the levels of BPDE-DNA adducts at the Monitoring Group (p=0.43).

 

These findings are in agreement with those of with (Pavanello et al. 1999[13]; van Schooten et al., 1992[15]). However, other studies found significant correlation between the levels of BPDE-DNA adducts and smoking in subjects with occupational exposure PAHs where exposure to tobacco products and PAHs acted synergistically to form BPDE-DNA adducts as reported by Rojas et al[16]. These discrepancy between these findings could be explained by inter-individual factor variability and route of exposure to PAHs which can play a major role in formation of BPDE-DNA adducts.

 

Our results suggests that importance of measurement of BPDE-DNA adducts and it's role as potential biomarker for exposure to PAHs. As it involves the DNA, it could be a surrogate marker to assess the risk for development of cancer [17,18]. Subjects who work closely with asphalt or at risk for exposure to PAHs should be monitored closely with frequent evaluation by physicians, laboratory testing and periodic assessment symptoms. In addition, hazardous materials should be assessed and labeled with the level of caution needed to handle the materials and with details of the protective equipment needed to handle them. Moreover, asphalt workers should use protective masks and gloves to avoid exposure to PAHs. Theworker should also be provided with educational sessions and pamphlets to explain these risks and detail methods of protection and decontamination after exposure. Finally, the temperature of the asphalt mixture should be lowered if possible to decrease the production of bitumen fumes and lower inhalation risk.

 

Given the known carcinogenic effect of B[a]P[1], the presence of BPDE-DNA adducts when measured in the WBC using ELISA technique correlates of the risk of development of B[a]P-induced diseases such as lung cancer]19]. Therefore, BPDE-DNA adducts can serve as a useful biomarker to assess prior exposure to PAHs and could potentially determine cancer risk [20].

 

CONCLUSIONS:

Our results highlight that importance of measurement of BPDE-DNA adducts and its role as potential biomarker for exposure to PAHs. As it involves the DNA, it could be a surrogate marker to assess the risk for development of cancer. Providing periodic medical evaluation and educational sessions, raising workers' awareness, using protective equipment and possibly lowering the temperature of the asphalt mixture can potentially lower the exposure risk to B[a]P and could potentially lower cancer risk.

 

ACKNOWLEDGEMENT:

Prof. MaroufAlkayer, Professor of Analytical Chemistry at Faculty of Pharmacy at Tishreen University in Syria.

 

CONFLICT OF INTEREST: No

 

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Accepted on 18.09.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(8): 3363-3366.