32P-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 shown to be genotoxic in in vitro test systems and induces tumors in the nasal 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 present study was to perform a similar investigation of other adducts of propylene 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 (32)P-postlabelling method involving nuclease P1 enhancement and radioisotope detector-coupled HPLC separation. All four adducts could be detected in DNA treated in vitro with propylene oxide. The sum of the levels of 1- and N(6)-HP-adenine amounted to 3.5% and the sum of 3-HP-cytosine and 3-HP-uracil to 1.7%, respectively, of 7-(2-hydroxypropyl)guanine (7-HP-guanine). In male Fischer 344 rats exposed to 500 p.p.m. propylene oxide by inhalation for 20 days, 1-HP-adenine was detected in all analysed tissues, including nasal epithelium, lung 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 induction in the rat following inhalation of propylene oxide. The levels of this adduct in the lung and in the lymphocytes were considerably lower, amounting to 15 and 9%, respectively, of that of the respiratory nasal epithelium. In rats killed 3 days after cessation of exposure, practically no decrease in 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-exposed 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 repair 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 probably not involved. The level of 1-HP-adenine in the propylene oxide-exposed rats was approximately 50 times lower than that of 7-HP-guanine. Nevertheless, this adduct is conveniently analysed and has high chemical stability and recovery, which results in high sensitivity (detection limit 0.3 mol/10(9) mol of normal nucleotides using 10 microgram DNA). 1-HP-adenine might, therefore, be considered as an alternative to 7-HP-guanine for monitoring exposure to propylene oxide.
Plna
K
K
Center for Nutrition and Toxicology, Department of Biosciences, Karolinska Institute, Novum, S-141 57 Huddinge, Sweden. kamila.plna@cnt.ki.se
Nilsson
R
R
Koskinen
M
M
Segerbäck
D
D
D000225:Adenine / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D002460:Cell Line; D003596:Cytosine / Q000378:metabolism*; D018736:DNA Adducts; D004260:DNA Repair; D004852:Epoxy Compounds / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009297:Nasal Mucosa / Q000187:drug effects / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D051381:Rats; D011916:Rats, Inbred F344; D014498:Uracil / Q000378:metabolism*
D002250:Carbon Radioisotopes; D018736:DNA Adducts; D004852:Epoxy Compounds; D010761:Phosphorus Radioisotopes; D014498:Uracil; D003596:Cytosine; D000225:Adenine; C009068:propylene oxide