Adduct Spots Research Articles

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

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.

32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female Sprague-Dawley rats.

The 32P-postlabeling technique was used to qualitatively establish the pattern of DNA adduct formation in mammary tissue and liver following administration of 1-nitropyrene to female Sprague-Dawley rats. 1-Nitropyrene (100 mg/kg b.w.) was administered by gavage in trioctanoin and the rats were sacrificed 24 h later. DNA was isolated from mammary fat pads and liver, enzymatically hydrolyzed to deoxyribonucleoside-3'-monophosphates and then converted to [5'-32P]3',5'-bisphosphates. The polyethyleneimine-cellulose (PEI-cellulose) TLC 32P-fingerprints revealed the presence of multiple putative adducts in the mammary fat pads and in the livers. To investigate the role of nitroreduction in the formation of these adducts, calf thymus DNA was incubated with [3H]1-nitropyrene in vitro in the presence of xanthine oxidase. The DNA was isolated and analyzed by the 32P-postlabeling technique. A major adduct spot was detected and confirmed as N-(deoxyguanosin-8-yl)-1-aminopyrene. This adduct cochromatographed with a minor in vivo adduct of DNA obtained from mammary fat pads and livers. However, the major adducts detected in vivo did not appear to originate from simple nitroreduction of 1-nitropyrene. The results of this study suggest that other metabolic pathways, such as ring oxidation, or ring oxidation followed by nitroreduction, may be responsible for the putative 1-nitropyrene-DNA adducts observed in mammary fat pads and livers of female Sprague-Dawley rats.

Evaluation of DNA damage in the oral mucosa of tobacco users and non-users by 32P-adduct assay.

Tobacco and its combustion products contain several known or potential human carcinogens and studies are now beginning to emerge for detecting DNA and protein adducts in tobacco users. A highly sensitive 32P-adduct assay, capable of measuring a wide spectra of aromatic and/or hydrophobic DNA adducts, was used to evaluate tobacco use-related adducts in human oral mucosal cells. Two volunteer groups of people participated: one, a random group of cigarette smokers (n = 28) with non-smoker controls (n = 13) from our Institute, and the other, a fisherfolk community of tobacco chewers (n = 19) with appropriate non-chewer controls (n = 15) from Bombay. The cells were dislodged from the oral cavity with either a cotton swab or a toothbrush. One to as many as 16 adduct spots in the range of 5-200 amol/micrograms DNA were detected in tobacco users as well as non-users. No adduct spot was detected in the tobacco users' samples that was not present in the controls, suggesting that tobacco use may not be responsible for the formation of these DNA adducts detected in the oral mucosa. The chromatographic behavior of the oral mucosal DNA adducts by selective PEI--cellulose TLC and their extractability in 1-butanol suggest that they have an aromatic and/or hydrophobic moiety and two of the major adducts have been tentatively assigned as aromatic amine derivatives. We have discussed various possibilities of endogenous and/or exogenous factors being involved in the formation of these adducts.

Comparison of 32P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz[a]anthracene--DNA adducts.

[3H]7,12-Dimethylbenz[a]anthracene-modified DNA obtained from mouse cells in culture was enzymatically hydrolyzed to nucleoside 3'-phosphates, postlabeled with [32P]phosphate, and the carcinogen-modified nucleoside bisphosphates were separated by thin layer chromatography. Each adduct spot was eluted, dephosphorylated and the resulting [3H]nucleoside adducts were analyzed by high pressure liquid chromatography so that the structural information available for the liquid chromatographic peaks could be applied to the spots obtained from the postlabeling procedure. After this cross referencing, specific dihydrodiol epoxide-nucleotide adducts can now be monitored by the postlabeling technique.

Further metabolism of diol-epoxides of chrysene and dibenz[a,c]anthracene to DNA binding species as evidenced by 32P-postlabelling analysis.

Incubation of r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide), the bay-region diol-epoxide of chrysene, with rat liver microsomes in the presence of NADP+ and DNA, followed by 32P-postlabelling analysis of the DNA, revealed the presence of at least two adducts not detected when anti-chrysene-1,2-diol 3,4-oxide was incubated with DNA alone. The formation of these adducts was not blocked by the epoxide hydrolase inhibitor 1,1,1-trichloropropane-2,3-oxide. One of the adducts cochromatographed with the adduct spot obtained when authentic 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide) was reacted with DNA. Evidence suggested that a second adduct could also be formed by further metabolism of anti-9-OH-chrysene-1,2-diol 3,4-oxide. In addition, evidence was obtained for the further metabolism of the syn-isomer of chrysene 1,2-diol 3,4-oxide and the anti-isomer of a non-bay-region diol-epoxide of dibenz[a,c]anthracene to DNA binding species, but not for that of either the anti- or syn-isomers of the bay-region diol-epoxide of benzo[a]pyrene, the anti-isomers of the bay-region or a non-bay-region diol-epoxide of benz[a]anthracene, or the anti-isomer of the bay-region diol-epoxide of benzo[b]fluoranthene.

Postlabeling analysis of carcinogen-DNA adducts in mussel, Mytilus galloprovincialis

The 32P-postlabeling analysis of DNA adducts formed in mussel digestive gland homogenates incubated with 2-aminofluorene (32 μ m) showed one major and one minor adduct in the range of 1 adduct per 1–4 × 10 8 nucleotides. The predominant adduct was found to be chromatographically similar to the known C8-aminofluorene-substituted guanine adduct. The metabolic activation of 2-aminofluorene was blocked in the presence of methimazole (470 μ m), a competitive inhibitor of FAD-containing monooxygenase. In contrast to 2-aminofluorene, benzo( a)pyrene-modified DNA either did not show any adduct or a very weak adduct spot (1 adduct per 1–4 × 10 9 nucleotides). The samples of DNA from mussel, carp and bream, which had no obvious exposure to pollutants, showed one to several weak ‘natural adducts’. The origin of these adducts is as yet undetermined. Since postlabeling assay enables measurement of carcinogen-DNA adducts formed with any, even unknown, chemicals from the composite environmental mixture of genotoxic pollutants, it may become a valuable tool for risk assessment also in marine environments.