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DNA Adducts

Q: What are the different types or variations of DNA adducts?

DNA adducts can be classified based on their chemical structure, origin, or the specific DNA bases they modify. Some common types include bulky DNA adducts, oxidative DNA adducts, and alkylated DNA adducts. Each type of adduct can have unique effects on DNA and may require different analytical techniques for detection and quantification.

Most cited protocols related to «DNA Adducts»

DNA Adduct Quantification in Carcinogen-Treated Cells

DNA was isolated from the cellular pellets by a chloroform/phenol extraction method (50 (link)). The amount of DNA recovered from each carcinogen treatment was determined by UV spectroscopy, assuming a concentration of DNA (50 μg/mL) is equal to 1.0 absorbance unit at 260 nm. Isotopically labeled internal standards of each DNA adduct were added to the DNA recovered from the treated hepatocytes at a level of 1 adduct per 10⁶ DNA bases. The DNA from the respective time points of the individually treated 4-ABP and HAA hepatocyte samples were then pooled. The enzymatic digestion conditions used for the hydrolysis of DNA (~2 – 10 μg) in 5 mM Bis-Tris-HCl buffer (pH 7.1, 50 μL) employed DNAse I for 1.5 h, followed by incubation with nuclease P1 for 3 h, and then digested with alkaline phosphatase and phosphodiesterase for 18 h (45 (link)). These enzyme digestion conditions were shown to be highly efficient in the recovery of the dG-C8 adducts of PhIP, MeIQx, IQ, and 4-ABP from calf thymus DNA modified with these carcinogens (42 (link)–45 (link),51 (link),52 (link)). The DNA adducts were purified by solid phase extraction, using HyperSep^™ filter SpinTips, as previously described (52 (link),53 (link)).

Nauwelaers G., Bessette E.E., Gu D., Tang Y., Rageul J., Fessard V., Yuan J.M., Yu M.C., Langouët S, & Turesky R.J. (2011). DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes. Chemical research in toxicology, 24(6), 913-925.

Publication 2011

2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline Alkaline Phosphatase calf thymus DNA Carcinogens Cells Chloroform Deoxyribonuclease I Digestive System Disorders DNA, A-Form DNA Adducts Enzymes Hepatocyte Hydrolases, Phosphoric Diester Hydrolysis N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine NOS2A protein, human nuclease H Pellets, Drug Phenol Solid Phase Extraction Spectrum Analysis Tromethamine

Capillary LC-MS/MS Quantification of DNA Adducts

The DNA adduct analyses were conducted with an Agilent 1100 Series capillary LC system (Agilent Technologies, Palo Alto, CA) equipped with an Aquasil C18 column (0.32 × 250 mm) from Thermo Fisher (Bellafonte, PA). Samples (2 μL) were injected, and analytes were separated with a gradient. The solvent conditions were held at 100% A (solvent composition: 0.01% HCO₂H and 10% CH₃CN) for 2 min, followed by a linear gradient to 100% B (solvent composition: 95% CH₃CN containing 0.01% HCO₂H) over 30 min at a flow rate of 6 μL/min. The MS instrumentation was a linear quadrupole ion trap mass spectrometer (LTQ, Thermo Fisher, San Jose, CA), and Xcalibur version 2.07 software was used for data manipulations. Analyses were conducted in the positive ionization mode and employed an Advance CaptiveSpray^™ source from Michrom Bioresource Inc. (Auburn, CA). Representative optimized instrument tuning parameters were as follows: capillary temperature 220 °C; source spray voltage 1.5 kV; source current 0.3 μA; no sheath gas, sweep gas or auxiliary gas was employed; capillary voltage 32 V; tube lens voltage 110 V; and in-source fragmentation 10 V.
The LTQ MS was employed in the tandem MS/MS scan mode to monitor the loss of deoxyribose from the protonated molecules of the adducts ([M+H-116]⁺), followed by the consecutive reaction monitoring scan mode at the MS³ scan stage, to characterize the product ions of the aglycone adducts [BH₂]⁺ (45 (link),53 (link),55 (link)). The total ion counts produced at the MS³ scan stage were used for quantitative measurements for all of the adducts except for dG-C8-IQ and [¹³C₁₀]-dG-C8-IQ. The ions monitored in MS → MS² → scan modes were as follows: dG-C8-PhIP (m/z 490.1 → 374.1); [¹³C₁₀]-dG-C8-PhIP (m/z 500.1 → 379.1); dG-C8-MeIQx (m/z 479.1 → 363.1); dG-C8-[²H₃C]-C8-MeIQx (m/z 482.1 → 366.1); dG-AαC (m/z 449.1 → 333.1); [¹³C₁₀]-dG-AαC (m/z 459.1 → 338.1); dG-C8-4-ABP (m/z 435.1 → 319.1); [¹³C₁₀]-dG-C8-4-ABP (m/z 445.1 → 324.1), dG-C8-IQ (m/z 464.1 → 348.1); [¹³C₁₀]-dG-C8-IQ (m/z 474.1 → 353.1). Isobaric interferences precluded the use of total ion counts for measurement of dG-C8-IQ and ([¹³C₁₀]-dG-C8-IQ; the extracted ions at m/z 302.2 and 331.1, and 307.2 and 336.2, respectively, produced at the MS³ scan stage, were used for quantitative measurements. The [¹³C₁₀]-dG-C8-4-ABP, [¹³C₁₀]-dG-C8-IQ and [¹³C₁₀]-dG-C8-MeIQx were employed as internal standards to estimate the levels of dG-N²-N⁴-ABP, dG-N²-IQ and dG-N²-MeIQx.
The normalized collision energies were set at 32 and 40, and the isolation widths were set at 3.0 and 1.0 Da, respectively, for the MS² and MS³ scan modes, for all adducts. The activation Q was set at 0.35 and the activation time was 10 ms, for both scan modes. Helium was used as the collision damping gas in the ion trap and was set at a pressure of 1 mTorr. One μscan was used for data acquisition. The automatic gain control settings were full MS target 30,000 and MSⁿ target 10,000, and the maximum injection time was 10 ms.

Publication 2011

2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline aquasil ARID1A protein, human Capillaries Deoxyribose DNA Adducts Helium isolation Lens, Crystalline N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine Pressure Radionuclide Imaging Solvents Tandem Mass Spectrometry Z 338

Surface Modification of Carbon Black Nanoparticles

To understand how surface modification can change the interaction of CBNP with biological systems we used PAHs to modify the surface of a toxicologically well-defined CBNP, Printex^®90 (P90). This is characterized by a high surface area and has been widely used in toxicological studies, resulting in only minor toxic effects [1 (link), 33 (link), 34 (link)].
For modification of the P90 surface, we used benzo[a]pyrene (BaP) and 9-nitroanthracene (9NA). BaP was chosen because of the well characterized toxicity of its metabolites, which are known to induce ROS and DNA adducts [23 (link), 35 (link)–38 (link)]. BaP is known to induce Cyp1A1 and 1B1, which then metabolize BaP to toxic metabolites; this therefore allows monitoring of BaP activity and its biological effect [39 (link), 40 (link)]. In contrast, 9NA is a PAH that occurs during combustion, and is regarded as a low toxicity PAH, as predicted by the Ames test and human cell mutagenicity assay [41 (link)–43 (link)]. However, due to its nitro group other toxic mechanisms can occur induced by intermediates resulting from reduction of the nitro group [41 (link)]. As coating of a particle does not necessarily represent the situation found in nanoparticles that acquire PAH during synthesis, we also generated CBNP by acetylene combustion [44 ]. The resulting acetylene soot (AS) had a mixture of PAHs on the surface (AS-PAH). In the suspensions we used, AS-PAH had a slightly larger specific surface area, but similar aggregate size and ζ-potential compared to PAH-coated P90, The physicochemical parameters of the different particles were evaluated by a variety of analytic test methods (see Tables 1, 2 in the Results section and Additional files 1, 2).

Lindner K., Ströbele M., Schlick S., Webering S., Jenckel A., Kopf J., Danov O., Sewald K., Buj C., Creutzenberg O., Tillmann T., Pohlmann G., Ernst H., Ziemann C., Hüttmann G., Heine H., Bockhorn H., Hansen T., König P, & Fehrenbach H. (2017). Biological effects of carbon black nanoparticles are changed by surface coating with polycyclic aromatic hydrocarbons. Particle and Fibre Toxicology, 14, 8.

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Publication 2017

9-nitroanthracene Acetylene Anabolism Benzo(a)pyrene Biological Assay Biopharmaceuticals Cells Cytochrome P-450 CYP1A1 DNA Adducts Mineralocorticoid Excess Syndrome, Apparent Mutagens Polycyclic Hydrocarbons, Aromatic Soot

Oral Carcinogen Exposure in Mice

Female B6C3F1 mice (Jackson Laboratories, Bar Harbor, ME), 6 weeks of age, were used in this study. The bioassays were carried out in accordance with NIH Guide for the Care and Use of Laboratory Animals and were approved by Institutional Animal Care and Use Committee. Initially, short-term studies were performed to determine whether our HPLC-MS/MS method is sensitive enough to detect DNA adducts in vivo in the oral tissues of treated mice. A group of three mice was treated with 12 nmol of DB[a,l]PDE topically into the oral cavity, and sacrificed at 48 h after the treatment. Another group of three mice was treated with 240 nmol DB[a,l]P per day for 2 days, sacrificed at 24 h after the second carcinogen dose. Finally, we carried out a time-course study and mice were treated with 24 nmol DB[a,l]P topically into the oral cavity 3 times per week for 5 weeks. Six animals per group were sacrificed at 48 h, 1, 2 and 4 weeks after the last dose. At termination, mice were sacrificed by CO₂ asphyxiation; soft tissues of the oral cavity, including buccal mucosa, floor of the mouth as well as soft tissues attached to the hard palate, were collected and pooled together for DNA adduct analysis.

Zhang S.M., Chen K.M., Aliaga C., Sun Y.W., Lin J.M., Sharma A.K., Amin S, & El-Bayoumy K. (2011). Identification and Quantification of DNA Adducts in the Oral Tissues of Mice Treated with the Environmental Carcinogen Dibenzo[a,l]pyrene by HPLC-MS/MS. Chemical research in toxicology, 24(8), 1297-1303.

Publication 2011

Animals Animals, Laboratory Asphyxia Biological Assay Carcinogens DNA Adducts Females Hard Palate High-Performance Liquid Chromatographies Institutional Animal Care and Use Committees Mice, House Mucosa, Mouth Oral Cavity Sublingual Region Tandem Mass Spectrometry Tissues

NAC Supplementation in Mice

Male transgenic or wild-type littermates were randomized to ad lib drinking water supplemented with 40 mM NAC or control drinking water with 40 mM alanine at weaning, and remained on supplemented water until sacrifice. Water with NAC or alanine was pH adjusted to pH 7.4 with sodium hydroxide [59] (link) and fresh water was provided thrice weekly. Control drinking water was supplemented with 40 mM alanine, a hydrophillic amino acid, similar in structure to, but lacking the thiol group of cysteine. 40 mM NAC supplemented in this manner has been shown to result in a mean dose of 1 g of NAC per kg body weight per day in C57BL/6 mice, a dose that reduces the accumulation of DNA adducts in mice treated with various carcinogens [60] (link).

Clark J., Clore E.L., Zheng K., Adame A., Masliah E, & Simon D.K. (2010). Oral N-Acetyl-Cysteine Attenuates Loss of Dopaminergic Terminals in α-Synuclein Overexpressing Mice. PLoS ONE, 5(8), e12333.

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Publication 2010

Alanine Amino Acids Animals, Transgenic Body Weight Carcinogens Cysteine DNA Adducts Males Mice, Inbred C57BL Mice, Laboratory Sodium Hydroxide Sulfhydryl Compounds

Most recents protocols related to «DNA Adducts»

DNA Binding Interactions Study

UV absorption titration was employed to study the binding interactions of the complexes with DNA in a UV-vis spectrophotometer (Cary 100) using reference Phosphate Buffer. The emission spectra were recorded in Fluorolog 3-111 (Horiba JobinYvon) spectrophotometer by noting the changes of fluorescence intensities of the ethidium bromide-DNA adduct with gradual addition of the complexes.

Acharya P., Kuila A., Pramanik U., Hathwar V.R., Brandao P., Mukherjee S., Maity S., Maity T., Maity R, & Chandra Samanta B. (2023). Combined theoretical and experimental insights on DNA and BSA binding interactions of Cu(ii) and Ni(ii) complexes along with the DPPH method of antioxidant assay and cytotoxicity studies. RSC Advances, 13(11), 7632-7644.

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Publication 2023

Buffers DNA Adducts Ethidium Bromide Fluorescence Phosphates Titrimetry

DNA Adduct Screening Protocol

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Publication 2023

DNA Adducts Filtration Immune Tolerance Ions Pharmaceutical Preparations Retention (Psychology)

Targeted MS2 Analysis of DNA Adducts

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Publication 2023

DNA Adducts DNA Fragmentation Ions isolation MS 54

Assessing Environmental Epigenetics: DNA Methylation and Adducts

Standard descriptive analyses were used to evaluate the levels of global and specific-DNA methylation and the formation of bulky DNA adducts in the WBCs of the study population according to smoking habit, exposure status, working districts, and other study variables. Multivariate analyses were performed by fitting linear regression models adjusted by age (continuous), gender, and smoking habit (smokers and non-smokers). Means Difference (MD) and 95% Confidence Intervals (C.I.) were used to evaluate the effect of each level of predictor variables with respect to its reference level and to estimate the relationship of DNA methylation and bulky DNA adduct biomarker with air pollution and specific pollutants. A p-value of 0.05 was considered statistically significant. Data were analysed using SAS9.3 and SPSS 20.0 (IBM SPSS Statistics, New York, NY, USA).

Munnia A., Bollati V., Russo V., Ferrari L., Ceppi M., Bruzzone M., Dugheri S., Arcangeli G., Merlo F, & Peluso M. (2023). Traffic-Related Air Pollution and Ground-Level Ozone Associated Global DNA Hypomethylation and Bulky DNA Adduct Formation. International Journal of Molecular Sciences, 24(3), 2041.

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Publication 2023

Air Pollution Biological Markers Dietary Fiber DNA Adducts DNA Methylation Environmental Pollutants Leukocytes Non-Smokers

Quantifying BPDE-DNA Adducts in Villous Tissue

Genomic DNA was isolated from human villous tissues using Tissue/Cell Genomic DNA extraction and Purification Kit (K1442-100; BioVision). The levels of BPDE-DNA adducts were assessed using BPDE-DNA adduct ELISA kit (STA-357; Cell Biolabs). Briefly, DNA samples (

2 micrograms per milliliter

) were sonicated into fragments with 200–1,000 bp as determined by 1% agarose gel and then incubated with anti-BPDE antibody (dilution 1:1,000; 235601, Cell Biolabs) in 96-well plates for 2 h at room temperature. After washing with wash buffer (310806; Cell Biolabs), the secondary antibody (10902; Cell Biolabs) was added to each well for 1 h, 3,3′,5,5′-tetramethylbenzidine buffer was added, and then the mixture was incubated for 20 min at room temperature. After termination of reaction, the relative levels of BPDE-DNA adducts were determined by measuring the absorbance at

450 nanometers

using a microplate reader (VL0L00D0; Thermo Fisher Scientific), with the Reduced DNA Standard (235602; Cell Biolabs) as absorbance blank. The amount of BPDE-DNA adducts was quantified using a BPDE-DNA standard curve. The results were expressed as nanograms of BPDE-DNA adducts per microgram of DNA.

Dai M., Huang W., Huang X., Ma C., Wang R., Tian P., Chen W., Zhang Y., Mi C, & Zhang H. (2023). BPDE, the Migration and Invasion of Human Trophoblast Cells, and Occurrence of Miscarriage in Humans: Roles of a Novel lncRNA-HZ09. Environmental Health Perspectives, 131(1), 017009.

Publication 2023

3,3',5,5'-tetramethylbenzidine 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide Antibodies, Anti-Idiotypic benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA Buffers Cells DNA Adducts Enzyme-Linked Immunosorbent Assay Genome Homo sapiens Immunoglobulins Sepharose Technique, Dilution Tissues

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Micrococcal nuclease is a laboratory enzyme used to cleave DNA and RNA. It is a calcium-dependent endonuclease that non-specifically hydrolyzes the phosphodiester bonds in nucleic acids.

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What are DNA adducts and why are they important?

What are the different types or variations of DNA adducts?

What are some common analytical techniques used to study DNA adducts?

Researchers rely on various analytical techniques to detect and quantify DNA adducts, such as mass spectrometry, immunoassays, and DNA sequencing. These methods allow them to identify the specific types of adducts, measure their levels, and investigate their formation and biological consequences. The choice of technique depends on factors like the type of adduct, sample size, and desired level of sensitivity and specificity.

How can DNA adduct research be challenging?

One of the main challenges in DNA adducts research is ensuring the accuracy and reproducibility of the analytical methods used. Differences in sample preparation, instrument calibration, and data analysis can lead to inconsistent results across studies. Researchers often need to sift through a large number of published protocols to find the most effective approaches for their specific research goals.

How can PubCompare.ai assist with DNA adducts research?

PubCompare.ai's innovative AI-powered protocol comparison tool can enhance the accuracy and reproducibility of DNA adducts research. The platform allows researchers to effortlessly locate the best protocols from literature, pre-prints, and patents, while leveraging AI-driven comparisons to identify the most effective approaches. This can help researchers take their DNA adducts studies to new heights and advance their understanding of these important DNA modifications. The platform's AI-driven analysis can hghlight key differences in protocol effectiveness, enabling researchers to choose the best option for reproducibility and accuracy.

More about "DNA Adducts"

DNA adducts are covalent modifications of DNA molecules that can occur naturally or as a result of exposure to environmental agents, such as chemicals or radiation.
These adducts can alter the structure and function of DNA, potentially leading to genetic mutations and increased risk of disease.
Researchers studying DNA adducts rely on various analytical techniques, including the use of tools like Instant Imager, Micrococcal nuclease, and QIAamp DNA Mini Kit, to detect and quantify these modifications.
Understanding the formation and biological consequences of DNA adducts is crucial in the field of biomedical research and clinical practice.
Calf spleen phosphodiesterase and OxiSelect BPDE DNA Adduct ELISA Kit are among the tools used to analyze and measure DNA adducts, while Immobilon Western Chemiluminescent HRP Substrate and BPDE-DNA adduct ELISA kit provide additional analytical capabilities.
PubCompare.ai's innovative AI-powered protocol comparison tool can enhance the accuracy and reproducibility of DNA adduct research by effortllessly locating the best protocols from literature, pre-prints, and patents, while leveraging AI-driven comparisons to identify the most effective approaches.
This solution, which can be integrated with tools like Voyager-DE STR and Penicillin, can help researchers take their DNA adduct studies to new heights and advance our understanding of these important DNA modifications.
Additionally, the DNeasy Blood & Tissue Kit is a useful tool for extracting and purifying DNA samples, which is essential for DNA adduct research.
By combining the power of these various tools and techniques, researchers can gain deeper insights into the formation, consequences, and potential applications of DNA adducts, ultimately driving progress in the field of biomedical research and clinical practice.