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Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)--DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE--DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE--DNA adducts as low as 180 fM in the presence of I billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methytation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotexical and epigenetic analysis. 10.1021/ac102416f

Identification and quantification of chemical DNA modifications provide essential information on genomic DNA changes, for example, epigenetic modifications and abnormal DNA lesions. In this vein, it requires to digest genomic DNA strands into single nucleosides, facilitating the mass spectrometry analysis. However, rapid digestion of such supramacromolecule DNA of several millions Daltons (molecular weight) into single nucleosides remains very challenging. Here, we constructed an immobilized benzonase capillary bioreactor and further tandemly coupled with immobilized snake venom phosphodiesterase and alkaline phosphatase capillary bioreactor to form a novel three-enzyme cascade bioreactor (BenzoSAC bioreactor). In these constructions, the chosen enzymes were immobilized onto synthetic porous capillary silica monoliths. With the tailor-made porous structure and high immobilized capacity and high digestion rate of benzonase, genomic DNA of >99.5% can be digested into single nucleosides within only 10 min when passing through the BenzoSAC bioreactor by microinjection pump. In contrast, traditional digestion requires 8-24 h. By offline coupling this benzoSAC bioreactor with liquid chromatography-tandem mass spectrometry, we detected 5-hydroxymethylcytosine, a major oxidation product of the epigenetically crucial 5-methylcytosine, in genomic DNA isolated from ladder cancer (T24) cells. The newly synthesized BenzoSAC bioreactor and the proposed mass spectrometry detection are promising for fast identification and analysis of structural modifications in DNA.

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.

17α-Ethinylestradiol (EE2) is the representative of environmental estrogens. Although EE2 can interact with some engineered nanoparticles (NPs), little is known about the bioactivity of NP-associated EE2 in organisms. In this study, we investigated the combined effects of the co-exposed carboxyl-functionalized single-walled carbon nanotubes (cf-SWCNTs) and EE2 in the human breast adenocarcinoma cell line (MCF-7 cells), focusing on the cytotoxicity and bioactivity. There were no significant differences in mitochondrial activity, membrane damage, and cell apoptosis when exposed to cf-SWCNTs with and without adsorbed EE2. However, the bioactivity of adsorbed EE2 on cf-SWCNTs was significantly inhibited. The calculated effective concentration of EE2 in cultured cells showed that less than 0.2% of the total adsorbed EE2 was released, indicating that most EE2 was retained on the cf-SWCNTs during cellular exposure. Furthermore, there were no obvious changes in the bioactivity of adsorbed EE2 in the culture medium containing 5-20% fetal bovine serum (FBS), even up to 10 days of incubation, indicating that the adsorbed EE2 on cf-SWCNTs is highly stable in the cell culture medium. These results mark a promising possibility for EE2 to be adsorbed by cf-SWCNTs in environmentally relevant settings and thereby influenced its toxicity and biological fate. This is also tempting for future studies involving risk assessment ways for association between NPs and contaminants in the environment.

Polyvinyl pyrrolidone polymer (PVP) has been widely applied in biological and medical fields. A few in vitro studies indicated that PVP might cause toxicity. However, the underlying mechanism is poorly understood. In this work, we found that PVP directly induced strand breakages of various DNA molecules, implicating a cleavage activity. Moreover, reactive oxygen species (ROS) scavenging analysis shows that DNA cleavage activity of PVP is not related to ROS-induced oxidation. As revealed by gel electrophoresis and liquid chromatography/mass spectrometry analysis, the major cleavage products of DNA were identified as two purine bases, guanine and adenine, suggesting that PVPs have a novel depurination activity. The selective depurination and DNA cleavage activity of PVPs were further confirmed by studying the interaction of PVP with four nucleosides and four well-designed oligodeoxynucleotides probes containing specific nucleotides. This study may provide insights into PVP-DNA interactions and resultant genotoxicity and may also open a new way for DNA study.

The toxicity of NPs is not well characterized in terms of their size. In particular, the size-based toxicity of fullerene (C(60)) remains an issue because of a lack of C(60) NPs with a well-controlled size. In this work, six fractions of the nano-C(60) aggregates (nC(60)) with different size distribution were prepared by a simple differential centrifugation. By using these nC(60) fractions, we demonstrate the size-dependent inhibition of DNA polymerase and reduced-size enhanced cytotoxicity. Above all, we found that nC(60) NPs with smaller size may have higher toxicity potency. These size-dependent effects were observed at the high exposure doses (4-6 mg/L). Interestingly, at 20-times lower and noncytotoxic doses, the size-dependent effect can be indicated by apoptosis-related fluorescent protein fused Bax translocation. Considering the toxicity of NPs is often ignored in the traditional end-point analysis for cytotoxicity when the exposure dose is low, the findings presented here will assist in the evaluation of the size-dependent cytotoxicity and dose-response relationships of toxicity mediated by nC(60) NPs at low doses.

A capillary monolithic bioreactor of snake venom phosphodiesterase (SVP) was constructed to generate different single-nucleotide mass ladders of oligodeoxynucleotides for mass spectrometry (MS)-based sequencing by immobilization. The immobilization of SVP in the porous silica monolith significantly enhances its stability for prolonged and repeated applications. The constructed capillary bioreactor has the advantages of handling (sub)microliter DNA samples and having good permeability. Benefiting from its good permeability, DNA solutions can be directly injected into the sequential digestion bioreactor simply by hand pushing or a low-pressure microinjection pump. Moreover, the immobilization of SVP facilitates the elimination or repression of the metal adducts of oligodeoxynucleotides, improving the analytical performance of MS sequencing. By the application of capillary bioreactor of immobilized SVP, the sequence-specific modification of single-stranded oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr) was identified, demonstrating its promising applications in identification of sequence-specific damage, which may further our understanding of DNA damage caused mutagenesis.