Detection of benzo[a]pyrene diol epoxide-DNA adducts in human placenta

Human placenta is a readily available organ that responds to maternal environmental insult and has been previously used to investigate metabolism and bioactivation of procarcinogens, for example, benzo[a]pyrene. HPLC in combination with synchronous fluorescence spectroscopy was used to examine 28 placentas for the presence of benzo[a]pyrene diol epoxide-DNA adducts, and 10 of these were found to be positive. DNA samples from these placentas were subsequently pooled and subjected to partial enzymatic digestion to oligonucleotide fragments. Concentration of those DNA fragments containing benzo[a]pyrene diol epoxide-DNA adducts was achieved by immunoaffinity chromatography with polyclonal antibodies raised against these adducts. Column eluates were hydrolyzed under mild acid conditions and extracted with an organic solvent. The presence of benzo[a]pyrene-7,10/8,9-tetrahydrotetrol residues in the extracts was determined by HPLC and synchronous fluorescence spectroscopy and was confirmed by GC/MS. The results unequivocally confirm bioactivation and formation of DNA adducts from benzo[a]pyrene in human placenta in vivo and establish a methodological approach to direct measurement of carcinogen-DNA adducts that are formed as a result of human environmental exposure.