P-32-postlabelling of propylene oxide 1-and N-6-substituted adenine and 3-substituted cytosine/uracil: formation and persistence in vitro and in vivo

Propylene oxide, a widely used monofunctional alkylating agent, has been sh own to be genotoxic in in vitro test systems and induces tumors in the nasa l tissues of experimental animals. Propylene oxide, like related alkylating agents, forms several different adducts with DNA bases, but predominantly at the 7-position of guanine. We have previously described the in vitro and in vivo formation and stability of this major adduct. The aim of the prese nt study was to perform a similar investigation of other adducts of propyle ne oxide, 1-(2-Hydroxypropyl)adenine (1-HP-adenine) and 3-(2-hydroxypropyl) cytosine (3-HP-cytosine), as well as their rearrangement products to N-6-(2 -hydroxypropyl)adenine (N-6-HP-adenine) and 3-(2-hydroxypropyl)uracil (3-HP -uracil), respectively, were analysed by a very sensitive P-32-postlabellin g method involving nuclease P1 enhancement and radioisotope detector-couple d HPLC separation. All four adducts could be detected in DNA treated in vit ro with propylene oxide. The sum of the levels of 1- and N-6-HP-adenine amo unted to 3.5% and the sum of 3-HP-cytosine and 3-HP-uracil to 1.7%, respect ively, of 7-(2-hydroxypropyl)guanine (7-HP-guanine). In male Fischer 344 ra ts exposed to 500 p.p.m. propylene oxide by inhalation for 20 days, 1-HP-ad enine was detected in all analysed tissues, including nasal epithelium, lun g and lymphocytes, whereas N-6-HP-adenine was only found in the tissues of the nasal cavities. The highest level of 1-HP-adenine (2.0 mol/10(6) mol of normal nucleotides, i.e. 2% of 7-HP-guanine) was found in the respiratory nasal epithelium, which also represents the major target for tumour inducti on in the rat following inhalation of propylene oxide. The levels of this a dduct in the lung and in the lymphocytes were considerably lower, amounting to 15 and 9%, respectively, of that of the respiratory nasal epithelium. I n rats killed 3 days after cessation of exposure, practically no decrease i n 1-HP-adenine was observed, indicating no or very slow repair. 3-HP-uracil could only be detected in the respiratory nasal epithelia of propylene-exp osed rats and its concentration was as low as 0.02 mol/10(6) mol of normal nucleotides (0.02% of 7-HP-guanine). Since 3-HP-uracil was chemically much more stable than the latter, the obtained animal data suggest repair of the cytosine and/or uracil adducts. Incubation of propylene oxide-reacted DNA with a protein extract from mammalian cells indicated that an enzymatic rep air mechanism exists for removal of 3-HP-cytosine, but not for 3-HP-uracil or 1- and N-6-HP-adenine. Another finding was that uracil glycosylase is pr obably not involved. The level of 1-HP-adenine in the propylene oxide-expos ed rats was similar to 50 times lower than that of 7-HP-guanine. Neverthele ss, this adduct is conveniently analysed and has high chemical stability an d recovery, which results in high sensitivity (detection limit 0.3 mol/10(9 ) mol of normal nucleotides using 10 mu g DNA). 1-HP-adenine might, therefo re, be considered as an alternative to 7-HP-guanine for monitoring exposure to propylene oxide.