**2.3 Determination of BPDE-DNA adduct levels**

After DNA was isolated from WBC, the samples were subsequently diluted to a concentration of 2 g 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. P value of <0.05 was considered to be statistically significant. Mann–Whitney U test was used to compare continuous data with nonnormal distribution, χ2 test was used to compare categorical variables between the two groups, and spearman coefficient and independent T- student tests were used.
