Detection of 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal after gavage of trans-4-hydroxy-2-nonenal or induction of lipid peroxidation with carbon tetrachloride in F344 rats.
The 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal (HNE-dGp-adducts) were quantitated in tissues of rats treated with trans-4-hydroxy-2-nonenal (HNE) or carbon tetrachloride, respectively, using a 32P-postlabeling method. The method development was based on chemically synthesized HNE-1,N2-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by Nuclease P1. They were subsequently reacted with gamma-32P-ATP to give the respective 3'-5'-bisphosphates, which were two-directionally separated on PEI-cellulose-TLC and quantitated by autoradiography. The labeling efficiency for the adduct standard was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. Internal standard was used to eliminate methodological variations. The determination of the limit of quantitation in DNA from rat tissues by spiking of HNE-dGp-adduct standard revealed a sensitivity of about 20 HNE-dGp-adducts/10(9) normal nucleotides. Background levels of HNE-dGp-adducts in tissues of rats including liver, kidney, lung, colon and forestomach were found in the range of 18-158 adducts/10(9) nucleotides with relatively high adduct levels in the liver and low adduct levels in kidney, lung and colon. These background levels were statistically significantly increased by the factor of 2 in liver, lung, colon and forestomach after induction of lipid peroxidation by carbon tetrachloride. The finding that background HNE-dGp-adduct levels may be in context with different metabolic activities of the tissues and the increase of HNE-dGp-adduct levels after application of carbon tetrachloride indicate that HNE-dGp-adducts are an endogenous lesion and that they are probably formed from radical initiated lipid peroxidation.
Wacker
M
M
Department of Toxicology, University of Würzburg, Versbacher Str. 9, D-97078, Würzburg, Germany.
Wanek
P
P
Eder
E
E
D000284:Administration, Oral; D000447:Aldehydes / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D002251:Carbon Tetrachloride / Q000008:administration & dosage / Q000633:toxicity*; D002855:Chromatography, Thin Layer; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism*; D005260:Female; D007274:Injections, Intraperitoneal; D015227:Lipid Peroxidation / Q000187:drug effects*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051381:Rats; D011916:Rats, Inbred F344; D012680:Sensitivity and Specificity
D000447:Aldehydes; D018736:DNA Adducts; C060934:1,N(2)-propanodeoxyguanosine; D004247:DNA; D002251:Carbon Tetrachloride; D003849:Deoxyguanosine; C027576:4-hydroxy-2-nonenal