Oxidatively DNA damaged and formalin-fixation procedures

Formaldehyde is the most commonly used fixative for the preparation of formalin-fixed paraffinembedded tissues (FFPETs) in the “reduction rooms” of pathology wards. Therefore, we analysed for the generation of 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one adducts (M1dG), an exocyclic DNA adduct considered to be a biomarker of oxidative stress and lipid peroxidation, in DNA extracted from the FFPETs of six liver and lung of C57BL/6 control mice and from the FFPETs of four cancer patients in respect to paired flash-frozen tissues using 32P-postlabeling. When the experimental animals were examined, the percentage of M1dG adducts was about 4-6 fold greater with the FFPET mouse samples as compared to flash-frozen mouse samples. Specifically, 4.75 M1dG ± 0.21 (SE) per 106 normal nucleotides (nn) were detected in the FFPET liver samples, and 1.07 M1dG ± 0.08 (SE) per 106 nn in the flash-frozen liver(p=0.02). Then, 3.80 M1dG ± 0.73 (SE) per 106 nn were measured in the FFPET lung samples, and 1.02 M1dG ± 0.07 (SE) per 106 nn in the flash-frozen lung (p=0.02). Also, significantly increased levels of oxidatively damaged DNA were detected in the human colon DNA from the FFPETs in respect to the flash-frozen tissues. There were 30.2 M1dG ± 7.7 (SE) per 108 nn in the colon mucosa DNA from the FFPETs and 4.4 M1dG ± 0.7 (SE) per 108 nn in the corresponding flash-frozen human tissues (p=0.016). Formalin penetration through cell membrane components induces excess oxidative stress, causing both direct oxidation in DNA and increased lipid peroxidation, which in turn produces M1dG adducts, a kind of DNA damage that can partially block DNA synthesis and induce error prone translesion synthesis. Excess of exocyclic DNA adducts in formalin-fixed specimens can stall DNA polymerases and contribute to the induction of artefactual sequence alterations during PRC amplification.