Immuno-slot-blot: A highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA

Nehls, Peter; Adamkiewicz, Jürgen; Rajewsky, Manfred F.

doi:10.1007/bf00390969

Cited by 64 publications

(39 citation statements)

References 9 publications

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“…These mechanisms comprise antioxidant systems for the deactivation of ROS molecules and efficient repair proteins for the elimination of oxidative DNA damage (19)(20)(21)(22)(23) (24)(25)(26)(27). These techniques can be applied for the determination of any DNA modification for which an appropriate monoclonal or polyclonal antibody is available.…”

Section: Introductionmentioning

confidence: 99%

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Nehls

Seiler

Rehn

et al. 1997

Environ Health Perspect

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Exposure of rats to quartz (or various other particles) can lead to the development of lung tumors. At the moment, the mechanisms involved in particle-induced tumor formation are not clarified. However, it is suggested that inflammation, in conjunction with the production of reactive oxygen species (ROS) and an enhancement of epithelial cell proliferation, may play a key role in the development of lung tumors. ROS induces 8-oxoguanine (8-oxoGua) and other mutagenic DNA oxidation products, which can be converted to mutations in proliferating cells. Mutation formation in cancer-related genes is a critical event with respect to tumor formation. In this study we investigated the effects of quartz (DQ1 2) and of the nontumorigenic dust corundum on the induction of 8-oxoGua in the DNA of rat lung cells, as well as on cell proliferation and pulmonary inflammation. Wistar rats were exposed by intratracheal instillation to quartz (2.5 mg/rat) or corundum (2.5 mg/rat) suspended in physiological saline; control animals exposed to physiological saline or left untreated. Measurements were carried out 7, 21, and 90 days after the exposures. 8-oxoGua levels were determined in lung tissue sections at the single cell level by immunocytological assay using a rabbit anti-8-oxoGua antibody. After exposure to quartz, 8-oxoGua levels were significantly increased at all time points of investigation. Additionally, we observed inflammation and an enhanced cell proliferation. Exposure to corundum had no adverse effects on the lung; neither increased 8-oxoGua levels nor enhanced cell proliferation or inflammation were detected. These observations support the suggestion that inflammation associated with increased 8-oxoGua levels in lung cells and increased cell proliferation is an important determinant for particle-induced development of lung tumors in the rat.

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Section: Introductionmentioning

confidence: 99%

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Nehls

Seiler

Rehn

et al. 1997

Environ Health Perspect

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“…DNA was dissolved in 10 mM Tris-HCl (pH = 8.0), quantified by UV spectrophotometry (Amersham Pharmacia Biotech), and stored at −80°C until assay. Semiquantification of ENU-induced O 6 -EtGua was carried out by an immunoslot blot method [13]. Briefly, 5 μg DNA was sonicated and then denatured at 100°C for 10 minutes.…”

Section: Immunoslot Blots Standard Curves and Dose Responsementioning

confidence: 99%

Polymorphisms of phase II xenobiotic-metabolizing and DNA repair genes and in vitro N-ethyl-N-nitrosourea-induced O6-ethylguanine levels in human lymphocytes

Jiao

Chang

Firozi

et al. 2007

Mutation Research/Genetic Toxicology and Environmental Mutagene

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This study tested the hypothesis that genetic variants of phase II detoxification enzymes and DNA repair proteins affect individual response to DNA damage from alkylating agents. In 171 healthy individuals, an immunoslot blot assay was used to measure O 6 -ethylguanosine (O 6 -EtGua) adduct levels in peripheral blood lymphocytes treated with N-ethyl-N-nitrosourea (ENU) in vitro. The genotypes of GSTM1, GSTT1, GSTP1 I 105 V and A 114 V, MGMT L 84 F and I 143 V, XPD D 312 N and K 751 Q, and XRCC3 T 241 M were determined. Demographic and exposure information was collected by in-person interview. Student's t test, analysis of (co)variance, and multiple linear regression models were used in statistical analyses. The mean and median (range) O 6 -EtGua levels were 94.6 and 84.8 (3.2-508.1) fmol/g DNA, respectively. The adduct level was significantly lower in people who smoked ≥ 25 years than that in never-smokers (square-root transformed mean values 8.20 versus 9.37, P = 0.03). Multiple linear regression models revealed that GSTT1 (β = −2.36, P = 0.009) polymorphism was a significant predictor of the level of adducts in 82 never-smokers, whereas the number of years smoked (β = −0.08, P = 0.005) and XRCC3 T 241 M (β = 2.22, P = 0.007) in 89 eversmokers. The association between GSTP1 I 105 V, MGMT I 143 V, and XPD D 312 N with the level of adducts was not conclusive. Each polymorphism could explain 2% to 10% of the variation of the adduct level. These observations suggest that GSTT1 null and XRCC3 T 241 M polymorphism may have some functional significance in modulating the level of ENU-induced DNA damage and these effects are smoking-dependent. Results from this exploratory study need to be confirmed in other experimental systems.

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“…This is true particularly for the analysis of human samples, where small amounts of sample and therefore DNA is normally available. The immunoslot blot (ISB) assay was first developed by Nehls and coworkers in 1984 (1). Since then the assay has been applied to the detection of several DNA adducts formed both in vitro (2)(3)(4)(5) and in vivo (6)(7)(8)(9)(10)(11)(12) and the use of both monoclonal (13,14) and polyclonal (2) antibodies has been described.…”

Section: Introductionmentioning

confidence: 99%

Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach

Moore

Xeniou

Zeng

et al. 2010

Analytical Biochemistry

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Immunoslot blot assays have been used for the analysis of many DNA adducts but problems are frequently encountered in achieving reproducible results. Each step of the assay has been systematically examined and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimisation was performed upon both the malondialdehyde-deoxyguanosine adduct (M 1 dG) and the O 6 -carboxymethyldeoxyguanosine adduct (O 6 CMdG) to demonstrate the applicability to other DNA adducts.Blood samples from the EPIC study (n = 162) were analysed for M 1 dG adducts and the data showed no correlation with adduct levels in other tissues indicating that the EPIC blood samples were not useful for studying M 1 dG adducts. Blood samples from a processed meat vs vegetarian diet intervention (n = 6) were analysed for O 6 CMdG and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole-genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 10 7 nucleotides for the analysis of O 6 CMdG.Subsequent reanalysis for O 6 CMdG showed a weakly significant increase in O 6 CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted.

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Immuno-slot-blot: A highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA

Cited by 64 publications

References 9 publications

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Polymorphisms of phase II xenobiotic-metabolizing and DNA repair genes and in vitro N-ethyl-N-nitrosourea-induced O6-ethylguanine levels in human lymphocytes

Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach

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