Tissue distribution of DNA adducts in CDF1 mice fed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-fquinoline (MeIQ)

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new 32P-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Development of a³²P-Postlabeling Method for the Detection of 1,N²-Propanodeoxyguanosine Adducts of 2-Hexenal in Vivo

Schüler

Budiawan

Eder

1999

“…A number of studies have been reported on HAA-DNA adduct formation and 32P-postlabeling analysis (17,19,22,(24)(25)(26)(27)(32)(33)(34)(35). There is considerable interlaboratory variation in the DNA adduct profiles, which appear quite complicated in many instances.…”

Section: Discussionmentioning

confidence: 99%

DNA Adduct Formation of the Food Carcinogen 2-Amino-3-methylimidazo[4,5-f]quinoline at the C-8 and N2 Atoms of Guanine

Turesky¹,

Markovic²

1994

DNA adduct formation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has been investigated by 32P-postlabeling. Similar adduct profiles were observed from calf thymus DNA modified in vitro with the putative carcinogenic metabolite N2-acetoxyamino-3-methylimidazo[4,5-f]-quinoline (N-acetoxy-IQ) and from hepatic DNA of rats treated with IQ. N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) accounted for approximately 90% of the total adducts observed in calf thymus DNA under postlabeling conditions where ATP was limiting; however, 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5- f]quinoline (dG-N2-IQ) was detected only when DNA was labeled with excess ATP. Under these labeling conditions, dG-C8-IQ and dG-N2-IQ accounted for approximately 75% and 7% of the total adducts, respectively. Five other spots accounted for the remaining radioactivity. Comparable results were obtained from rat liver DNA. Following DNA adduct enrichment by solid phase extraction, dG-C8-IQ and dG-N2-IQ accounted for 60-76% and 10-13%, respectively, of the total adducts in rat liver. The adduct profiles obtained from reaction of 2'-deoxyguanosine 3'-monophosphate (dG-3'-PO4-) with the photoactivated azide derivative of IQ, 2-azido-3-methylimidazo[4,5-f]quinoline (N3-IQ), were qualitatively similar to those obtained by reaction with N-acetoxy-IQ. The C-8 and N2 adducts were the only reaction products detected. The reactivity and sites of adduct substitution were dependent upon solvent conditions and pH, with increasing adduct formation under alkaline pH. The chemical reactivity of photoactivated N3-IQ with dG-3'-PO4- was significantly greater than that of N-acetoxy-IQ when reactions were conducted in water, in citrate buffer (pH 5.0), or in phosphate buffer (pH 7.4). Increased reactivity was attributed to increased levels of dG-C8-IQ adduct formation, except for reactions conducted in citrate buffer (pH 5.0), where there was a proportional increase in both C-8 and N2 guanine adducts. However, the chemical reactivity of these two IQ derivatives and their sites of dG substitution were identical when the reactions were conducted in phosphate buffer (pH 9.0). The ratio of the dG-N2-IQ adduct to the total adducts increased at alkaline pH in reactions involving N3-IQ, but the ratio was not affected by a change in the pH of the medium for reactions with N-acetoxy-IQ. The ratio of the dG-N2-IQ adduct to the total adducts also increased as a function of phosphate concentration for reactions involving both N-acetoxy-IQ and N3-IQ.(ABSTRACT TRUNCATED AT 400 WORDS)

“…It was concluded that the dG-N 2 -IQ adduct is not detected when labeling under adduct intensification conditions and that adduct enrichment before postlabeling is necessary for quantitative labeling of IQ-DNA adducts (30). Adduct enrichment by nuclease P1 (38) or butanol extraction (34) has been suggested to be unsuitable for some heterocyclic amine DNA adducts (21). In a few cases, however, the butanol extraction assay and/ or the nuclease P1 assay have been used for adduct enrichment of heterocyclic amine DNA adducts (18,39,40).…”

Section: Discussionmentioning

confidence: 99%

“…32 P-Postlabeling is a very sensitive technique which can detect DNA adducts in the range of 1 adduct per 10 7 -10 10 nucleotides in a 10 µg DNA sample. The ATP-deficient version of the 32 P-postlabeling assay followed by chromatographic separation by 32 P-TLC has been extensively used for analysis of DNA adducts formed by several different food mutagens in vitro as well as in rodents and monkeys (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). However, in order to detect all DNA adducts in a sample, it is necessary to perform 32 P-postlabeling with an excess of [ 32 P]ATP (30).…”

Section: Introductionmentioning

confidence: 99%

³²P-HPLC Analysis of DNA Adducts Formed in Vitro and in Vivo by 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline, Utilizing an Improved Adduct Enrichment Procedure

Wohlin¹,

Zeisig²,

Gustafsson

et al. 1996

DNA adducts of 2-amino-1 methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-diMeIQx), synthesized in vitro with calf thymus DNA and formed in vivo in the male Wistar rat, were enriched from digested DNA by butanol extraction before 32P-postlabeling. The recovery after butanol enrichment was 79% and 32% for in vitro modified PhIP- and 4,8-diMeIQx-DNA adducts, respectively. Crude postlabeling mixtures were chromatographically separated by high-performance liquid chromatography with on-line 32P-detection (32P-HPLC). The major PhIP- and 4,8-diMeIQx-DNA adducts formed in vitro cochromatographed with the respective pdGp-C8 adduct standard. 32P-HPLC was also used to separate hydrolysates of in vitro formed PhIP-DNA and 4,8-diMeIQx-DNA that had been 32P-postlabeled under ATP-deficient conditions. The adduct recovery of the ATP-deficient method relative to the improved butanol enrichment procedure was 29% and 59% for total PhIP-DNA and 4,8-diMeIQx-DNA adducts, respectively. Simplified DNA adduct patterns were obtained when the postlabeling mixtures were incubated with nuclease P1, suggesting incomplete DNA hydrolysis. After nuclease P1 treatment, the major DNA adducts of PhIP and 4,8-diMeIQx formed in vitro cochromatographed with the respective pdG-C8 adduct standard. In vivo PhIP formed what appeared to be multiple DNA adducts; however, after nuclease P1 treatment the PhIP-associated peaks were concentrated into a single peak cochromatographing with pdG-C8-PhIP, 4,8-diMeIQx formed multiple DNA adducts in vivo. Nuclease P1 treatment resulted in two 4,8-diMeIQx related peaks, one cochromatographing with pdG-C8-4,8-diMeIQx. The second peak remains unidentified. The improved workup procedures in combination with the high resolution and reproducibility of the 32P-HPLC system should be useful for characterization of PhIP- and 4,8-diMeIQx-DNA adducts in DNA modified by complex mixtures.