Detection of cyclic 1, N²-propanodeoxyguanosine adducts in DNA of rats treated with N-nitrosopyrrolidine and mice treated with crotonaldehyde

Abstract

Cyclic 1,N2-propanodeoxyguanosine adducts are formed in vitro in DNA treated with alpha-acetoxy-N-nitrosopyrrolidine or its metabolite, crotonaldehyde. However, the in vivo formation of these cyclic adducts in DNA has not been demonstrated due to the lack of a sensitive detection method. In this study, a 32P-postlabeling method specific for the detection of 1,N2-propanodeoxyguanosine adducts was developed by using the corresponding 3'-monophosphates as standards. This method was validated by using DNA modified in vitro. It was then applied for the in vivo experiments in which hepatic DNA of rats treated with N-nitosopyrrolidine (NPYR) (total dose, 1.0 mmol) in drinking water or skin DNA of Sencar mice treated topically with crotonaldehyde (1.4 mmol) was isolated and subjected to 32P-postlabeling analysis. 1,N2-Propanodeoxyguanosine adducts were detected in these DNA samples. The minimal levels of adducts from liver DNA and skin DNA detected were estimated to be approximately 0.06 and approximately 0.24 mumol/mol guanine respectively. Interestingly, a background adduct spot chromatographically indistinguishable from the 1,N2-cyclic adducts was observed in the liver DNA of untreated rats. However, no such background adduct was detected in skin DNA of mice. This method demonstrated for the first time the in vivo formation of the cyclic 1,N2-propanodeoxyguanosine adducts.

Analysis of Adducts in Hepatic DNA of Rats Treated with N-Nitrosopyrrolidine

“…We also obtained convincing evidence for the presence of N 6 -THF-dAdo (14) and N 4 -THF-dCyd (17) but quantitation was more difficult due to their low amounts and the presence of interfering peaks in some samples. Several lines of evidence support these conclusions.…”

“…A limitation of this study is the method of quantitation of N 2 -THF-dGuo (13), N 6 -THF-dAdo (14), and dThd adducts 15 and 16. While NaBH 3 CN treatment of DNA converts these adducts to the corresponding stable 4- (HOB)-adducts 18-21 Several previous studies of reactants other than α-acetoxyNPYR have demonstrated the in vitro formation of adducts resulting from the reaction of oxonium ions, lactols, or related intermediates with deoxyribonucleosides or DNA.…”

“…Two groups, each consisting of 4 rats weighing approximately 250 g each, were given either tap water or tap water containing 600 ppm NPYR for 1 week, then sacrificed. The same protocol was used previously for detection of NPYR-DNA adducts (14).…”

1,N 2-Deoxyguanosine Adducts of Acrolein, Crotonaldehyde, and trans-4-Hydroxynonenal Cross-link to Peptides via Schiff Base Linkage

“…This observation suggests that an analogous reaction of HNE with a protein lysine and a nucleic acid amine might represent a plausible pathway in the formation of DPCs. In addition, the detection of 1,N 2 -deoxyguanosine adducts of acrolein, crotonaldehyde, and 4-hydroxynonenal in human and rodent tissues, both as endogenous adducts and following chemical treatment (27)(28)(29)(41)(42)(43), may indicate that these stable DNA adducts exist as intermediates along a pathway of formation for DPCs. Consistent with such a mechanism, the results presented here demonstrate that peptide amines can react with these DNA adducts to form DNA-peptide cross-links via Schiff base linkage.…”

2‐Butenal (Crotonaldehyd) [MAK Value Documentation in German language, 2007]

“…In vitro wurden Proteinaddukte (Ichihashi et al 2001) und Histon-DNA-Crosslinks (Kuykendall und Bogdanffy 1992) durch Umsetzung mit 2-Butenal erzeugt. Nach Inkubation mit 2-Butenal ließen sich in isolierter Kalbsthymus-DNA, kultivierten CHO-Zellen und menschlichen Fibroblasten sowie lokal und systemisch in verschiedenen Geweben von in vivo behandelten Mäusen und Ratten zyklische 1,N 2 -Propanodesoxyguanosin-Addukte nachweisen, die als Folgeprodukte einer Michael-analogen Reaktion der aktivierten C-C-Doppelbindung des 2-Butenals mit dem exozyklischen Stickstoff des Desoxyguanosin-Rests aufzufassen sind (Budiawan und Eder 2000;Chung et al 1988Chung et al , 1989Eder et al 1999;Foiles et al 1990;Wilson et al 1991). Zyklische 1,N 2 -Propanodesoxyguanosin-Addukte werden in vivo in Tier und Mensch auch endogen gebildet (Chung et al 1999(Chung et al , 2003.…”