Detection by 32P-postlabelling of DNA adducts induced by free radicals and unsaturated aldehydes formed during the aerobic decomposition of fecapentaene-12.
Fecapentaene-12 (fec-12), excreted in human faeces, is genotoxic to human cells and a known animal carcinogen. The mechanism of its genotoxicity is unknown but may involve direct alkylation and/or free-radical generation. The formation of reactive species during fec-12 aerobic degradation was thus investigated by electron paramagnetic resonance (EPR) and NMR spectroscopic techniques. Oxy- and alkyl-radicals were detected as the 5,5'-dimethyl-1-pyrroline-N-oxide spin-trap adducts at fec-12 concentrations of between 0.1 and 2.0 mM. Under anaerobic conditions no free-radical generation was observed. NMR spectroscopy indicated that fec-12 degraded at least initially into three unsaturated aldehydes. The co-formation of free-radicals and unsaturated aldehydes suggests that fec-12 decomposed aerobically via a process analogous to lipid peroxidation. As both types of species, thus formed, may subsequently interact with DNA to form adducts, fec-12-induced DNA damage was investigated by 32P-postlabelling techniques. Using procedures that detect alkyl-type adducts, a number of putative adducts were detected in fec-12-treated DNA; two of similar mobility were observed in fec-12-treated 2'-deoxyguanosine-3'-monophosphate. Adducts with similar mobility have been detected in acrolein-treated DNA. One adduct with similar mobility was also observed in DNA obtained from normal human fibroblasts treated with fec-12. Using a C-18 ODS column, these putative adducts were eluted in 60-85% methanol, whereas 8-hydroxydeoxyguanosine-3'-monophosphate (8OHdGp) was eluted with 1% acetonitrile. Also unlike these putative adducts, the detection of 8OHdGp required HPLC fractionation prior to 32P-postlabelling. The formation of adducts, possibly aldehyde-related, and free-radical damage suggests that fec-12 genotoxicity may be the result of several different mechanisms, the relative importance of each is as yet unknown. Hydroxyl radicals were also detected during the aerobic decomposition of deca-2,4,6,8-tetraenal, a possible degradation product of fec-12 and a less potent mutagen, suggesting that free-radical generation may have only a minor role in fec-12-induced genotoxicity.
Povey
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AC
Laboratory of Human Carcinogenesis, NCI, NIH, Bethesda, MD 20892.
Wilson
V L
VL
Zweier
J L
JL
Kuppusamy
P
P
O'Neill
I K
IK
Harris
C C
CC
D001345:Autoradiography; D003497:Cyclic N-Oxides / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D006801:Humans; D009153:Mutagens / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011090:Polyenes / Q000378:metabolism*; D013113:Spin Labels
D003497:Cyclic N-Oxides; D003848:Deoxyguanine Nucleotides; D009153:Mutagens; D010761:Phosphorus Radioisotopes; D011090:Polyenes; D013113:Spin Labels; C069977:2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy; C017245:5,5-dimethyl-1-pyrroline-1-oxide; C007257:2'-deoxyguanosine 5'-phosphate; C037357:1-(1-glycero)dodeca-1,3,5,7,9-pentaene; D004247:DNA