Effect of tissue fixation on recovery of DNA adducts in the 32P-postlabelling assay

The suitability of fixed tissue as a source of DNA for 32P-postlabelling studies on DNA adduct formation has been investigated. Tissues (spleen, liver, lung, colon and kidney) from rats treated i.p. with benzo[a]pyrene (BaP) (100 mg/kg) or 2-acetylaminofluorene (AAF) (40 mg/kg) were removed and placed in formalin fixative for 1, 7, 28 or 92 days. DNA was isolated and 32P-postlabelled with nuclease P1 enhancement for BaP-modified DNA and with butanol enhancement for AAF-modified DNA. There was a marked loss of adducts with time of fixation, most noticeably between 1 and 7 days. DNA from tissue that had been fixed for 92 days and then wax-embedded had similar levels of adducts to the 92-day fixed-only samples. Further tissue samples were fixed for 24 h with formalin, modified Methacarn or Bouin's and then wax-embedded. DNA was then extracted either immediately or 28 days later. Adducts were recovered from formalin- and Methacarn-treated tissues at levels comparable to those detected in DNA from frozen tissues and levels were stable once the tissues were wax-embedded. However, DNA recovery from Bouin's-fixed tissue was poor. The results indicate that only tissues that have been wax-embedded after the minimum required fixation period are suitable sources of DNA for 32P-postlabelling analysis.