Polycyclic Aromatic Hydrocarbon-DNA (PAH-DNA) Adducts

PAH diol epoxide-DNA adducts were analyzed by competitive ELISA, using methods described previously (Divi et al., 2002). Briefly, 96 microwell plates coated with 2 ng of benzo(a)pyrene diol epoxide (BPDE)-I-DNA (5 adducts/10³ nucleotides) and rabbit antiserum #29 (Poirier 1980) was used with BPDE-DNA as a standard. DNA was isolated from frozen PBMC samples according to standard procedure using phenol/chloroform/isoamyl alcohol. DNA was assayed for PAH-DNA adducts after sonication and denaturation by laboratory technicians blinded to exposure status. For analytical purposes, those samples with <15% inhibition are considered non-detectable and assigned a value of 1 adduct /10⁸ nucleotides, an amount midway between the lowest positive value and zero. A 5% blinded duplication was carried out using those subjects with the most DNA available. As an additional quality control, a DNA sample from an animal treated with BP was also assayed was run with the sample batch.