Characterization of adducts formed in the reaction of 2-chloro-4-methylthiobutanoic acid with 2'-deoxyguanosine.
2-chloro-4-methylthiobutanoic acid (CMBA) is a direct-acting mutagen found in salt-nitrite-treated Sanma fish or similarly treated methionine solution. In this study, CMBA was reacted with 2'-deoxyguanosine (dG) in phosphate buffer (pH 7.4) at 37 degrees C. The HPLC-UV analysis showed that two products were mainly formed during the reaction. These were isolated, purified by semipreparative HPLC, and characterized as N7-guanine adducts: N7-(3-carboxy-3-methylthiopropyl)guanine (A1) and N7-(1-carboxy-3-methylthiopropyl)guanine (A2). Furthermore, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis was employed to investigate the possible formation of minor products during the time-course of the reaction of CMBA with dG. It was found that N7-dG adducts, the precursors of A1 and A2, were formed early in the reaction and that subsequently the spontaneous depurination occurred to yield stable N7-guanine adducts A1 and A2. Stability studies in phosphate buffer (pH 7.4) at 37 degrees C showed that the amount of each N7-dG adduct decreased rapidly with a half-life of 6 h and 4 h to yield A1/A2, respectively. A regioisomer of N7-dG adducts was also observed in the LC/ESI-MS/MS analysis, but it was not characterized in detail because it was present only in trace amounts. On the basis of structural features, A1 and A2 seemed to be formed from the reaction of dG with 1-methyl-2-thietaniumcarboxylic acid, an intermediate resulting from the cyclization of CMBA. However, A2 might also have formed from the direct reaction of dG and CMBA. N7-Alkylation of the guanine residue and subsequent depurination are known to produce apurinic sites in DNA that induce point mutations and may be responsible for the observed CMBA-induced mutagenesis.
Kimura
Sachiko
S
School of Human Science and Environment, University of Hyogo, Shinzaike, Himeji 670-0092, Japan. sachiko@shse.u-hyogo.ac.jp
Nakayama
Miki
M
Hatano
Tsutomu
T
Segawa
Ayako
A
Watanabe
Toshiaki
T
Hayatsu
Hikoya
H
Arimoto-Kobayashi
Sakae
S
D002087:Butyrates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000737:chemistry*; D003849:Deoxyguanosine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds
C093068:2-chloro-4-methylthiobutanoic acid; D002087:Butyrates; D018736:DNA Adducts; D013438:Sulfhydryl Compounds; D003849:Deoxyguanosine