Quantification of cyanobacterial cyclic di-guanosine monophosphate (c-di-GMP) by liquid chromatography electrospray ionization tandem mass spectrometry.

Cyclic di-guanosine monophosphate (c-di-GMP) is a second messenger found ubiquitously in bacteria. This signaling molecule regulates a variety of physiological activities such as phototaxis and flocculation in cyanobacteria and is critical for their environmental adaptation. Although genes encoding the enzymes for synthesis and/or degradation of c-di-GMP are found in the genomes of both multicellular and unicellular cyanobacteria, little is known about the biological functions of these enzymes in cyanobacterial cells. Here we have established a robust and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method for c-di-GMP quantification using a cost-effective solvent, methanol. Quantification methods were validated by measuring c-di-GMP in the cyanobacterium Synechococcus elongatus PCC 7942 through spiking and recovery assays after which the method was applied to examine short-term changes in cellular levels of c-di-GMP in response to a transition from light to dark or from dark to light in S. elongatus. Results showed that a transient increase in c-di-GMP upon transitioning from light to dark was occurring which resembled responses involving cyclic adenosine monophosphate and other second messengers in cyanobacteria. These findings demonstrated that our method enabled relatively specific and sensitive quantification of c-di-GMP in cyanobacteria at lower cost.

Characterization of N-(2,6-dimethylphenyl)hydroxylamine adducts of 2'-deoxyguanosine under weakly basic conditions.

Aromatic amines are a class of chemical carcinogens that are activated by cytochrome P450 enzymes to form arylhydroxylamines that are conjugated to form N-acetoxyarylamines or N-sulfonyloxyarylamines. These conjugates undergo N-O bond cleavage to become reactive nitrenium ions that may form DNA adducts. Numerous studies in the past using N-acetoxyarylamines to investigate DNA adduct formation were conducted, however, less is known in regard to DNA adduct formation directly from arylhydroxylamines - especially under conditions that mimic the physiological conditions of cells such as weakly basic conditions. In this study, 2'-deoxyguanosine (dG) was exposed to N-(2,6-dimethylphenyl)hydroxylamine (2,6-DMPHA) and N-phenylhydroxylamine (PHA) at pH 7.4 without enzymes and analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS). 2,6-DMPHA exposure resulted in the production of relatively low amounts of adducts however the identities of at least six different adducts that were formed through reactions with carbon, nitrogen and oxygen of 2'-deoxyguanosine were proposed based upon different analytical approaches including HRMS CID fragmentation and NMR analyses. Contrastively, PHA exposure under identical conditions resulted in one adduct at the C8 position. It was concluded from these results and results of theoretical calculations that nitrenium ions produced from 2,6-DMPHA were relatively more stable resulting in longer nitrenium ion lifetimes which ultimately led to greater potential for 2,6-DMPHA nitrenium ions to react with multiple sites on dG.

In vitro DNA/RNA Adductomics to Confirm DNA Damage Caused by Benzo[a]pyrene in the Hep G2 Cell Line.

In the development of new chemical substances, genetic toxicity evaluations are a high priority for safety risk management. Evaluation of the possibility of compound carcinogenicity with accuracy and at reasonable cost in the early stages of development by in vitro techniques is preferred. Currently, DNA damage-related in vitro genotoxicity tests are widely-used screening tools after which next generation toxicity testing may be applied to confirm DNA damage. DNA adductomics may be used to evaluate DNA damage in vitro, however confirmation of DNA adduct identities through comparison to authentic standards may be time-consuming and expensive processes. Considering this, a streamlined method for confirming putative DNA adducts that are detected by DNA adductomics may be useful. With this aim, in vitro DNA adductome methods in conjunction with in vitro RNA adductome methods may be proposed as a DNA adductome verification approach by which to eliminate false positive annotations. Such an approach was evaluated by conducting in vitro assays whereby Hep G2 cell lines that were exposed to or not exposed to benzo[a]pyrene were digested to their respective 2'-deoxynucleosides or ribonucleosides and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) by comparative DNA and RNA adductomics through neutral loss targeting of the [M + H]+ > [M + H - 116]+ or [M + H]+ > [M + H -132]+ transitions over predetermined ranges. Comparisons of DNA adductome maps revealed putative DNA adducts that were detected in exposed cells but not in unexposed cells. Similarly, comparisons of RNA adductome maps revealed putative RNA adducts in exposed cells but not in unexposed cells. Taken together these experiments revealed that analogous forms of putative damage had occurred in both DNA and RNA which supported that putative DNA adducts detected by DNA adductomics were DNA adducts. High resolution mass spectrometry (HRMS) was utilized to confirm that putative nucleic acid adducts detected in both DNA and RNA were derived from benzo[a]pyrene exposure and these putative adducts were identified as 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene- (B[a]PDE)-type adducts. Overall, this study demonstrates the usefulness of utilizing DNA/RNA adductomics to screen for nucleic acid damage.

Triple quadrupole mass spectrometry comparative DNA adductomics of Hep G2 cells following exposure to safrole.

A DNA adduct screening pipeline was constructed to apply triple quadrupole mass spectrometry comparative DNA adductomics to investigate the effects of the naturally-occurring plant constituent, safrole (4-allyl-1,2-methylenedioxybenzene), on human hepatoma cells, Hep G2. DNA from Hep G2 cells that were exposed to or not exposed to safrole were digested to 2'-deoxynucleosides and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) whereby the neutral loss of 2'-deoxyribose was targeted by monitoring the [M+H]+ > [M+H - 116]+ transition over a defined range. Comparative analyses through construction of DNA adductome maps revealed numerous putative DNA adduct candidates. Targeted product ion scan investigations allowed for detailed fragmentation ion analyses and the identities of at least five bulky alkylated adducts of 2'-deoxyguanosine and 2'-deoxyadenosine with molar masses greater than 400 Da each were proposed. All adducts were derived from safrole exposure and pathways to explain the occurrence of these adducts in Hep G2 cells through metabolism of safrole are discussed. This study demonstrates the potential utility of constructing triple quadrupole MS comparative DNA adductomics pipelines to screen chemicals for DNA adducts by using human cell lines.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galß1-4Glc). MytiLec revealed ß-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

Differential arsenic mobilization from As-bearing ferrihydrite by iron-respiring Shewanella strains with different arsenic-reducing activities.

Arsenic immobilization and release in the environment is significantly influenced by bacterial oxidation and reduction of arsenic and arsenic-bearing minerals. In this study, we tested three iron-reducing bacteria, Shewanella oneidensis MR-1, Shewanella sp. HN-41, and Shewanella putrefaciens 200, which have diverse arsenate-reducing activities with regard to reduction of an As-bearing ferrihydrite slurry. In the cultures of S. oneidensis MR-1 and Shewanella sp. HN-41, which are not capable of respiratory reduction of As(V) to As(III), arsenic was maintained predominantly in its pentavalent form, existing in particulate poorly crystalline As-bearing ferrihydrite and formed small quantities of a stable ferrous arsenate [Fe3(AsO4)2] precipitate. However, in the culture of the As(V) reducer, S. putrefaciens 200, As(V) was reduced to As(III) and a small fraction of As-bearing ferrihydrite was transformed into ribbon-shaped siderite that subsequently re-released arsenic into the liquid phase. Our results indicated that release of arsenic and formation of diverse secondary nanoscale Fe-As minerals are specifically closely related to the arsenic-reducing abilities of different bacteria. Therefore, bacterial arsenic reduction appears to significantly influence As mobilization in soils, minerals, and other Fe-rich environments.

A lectin from the mussel Mytilus galloprovincialis has a highly novel primary structure and induces glycan-mediated cytotoxicity of globotriaosylceramide-expressing lymphoma cells.

A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galß1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

Cytotoxicity and glycan-binding properties of an 18 kDa lectin isolated from the marine sponge Halichondria okadai.

A divalent cation-independent lectin-HOL-18, with cytotoxic activity against leukemia cells, was purified from a demosponge, Halichondria okadai. HOL-18 is a 72 kDa tetrameric lectin that consists of four non-covalently bonded 18 kDa subunits. Hemagglutination activity of the lectin was strongly inhibited by chitotriose (GlcNAcß1-4GlcNAcß1-4GlcNAc), fetuin and mucins from porcine stomach and bovine submaxillary gland. Lectin activity was stable at pH 4-12 and temperatures lower than 60 °C. Frontal affinity chromatography with 16 types of pyridylaminated oligosaccharides indicated that the lectin had an affinity for N-linked complex-type and sphingolipid-type oligosaccharides with N-acetylated hexosamines and neuramic acid at the non-reducing termini. The lectin killed Jurkat leukemia T cells and K562 erythroleukemia cells in a dose- and carbohydrate-dependent manner.

MRP1 expressed on Burkitt's lymphoma cells was depleted by catfish egg lectin through Gb3-glycosphingolipid and enhanced cytotoxic effect of drugs.

A novel anticancer mechanism of catfish (Silurus asotus) egg lectin (SAL) was found to occur via the down-regulation of the membrane transopter protein, MRP1 (multidrug resistance associate protein-1) on Burkitt's lymphoma cells through Gb3(Galα1-4Galß1-4Glc)-glycosphingolipid. Although SAL did not influence the viability of the cells directly, only 10 and 100 ng/mL of vincristine and etoposide, respectively induced anticancer effects when the lectin was applied in conjunction with these drugs. These phenomena were specifically inhibited by the co-presence of the α-galactoside, melibiose, which is a strong haptenic sugar of SAL that mimicks Gb3. The degree of expression regulation of the transporter proteins on the cells surface was investigated through the examination of the binding between SAL and Gb3-glycosphingolipid by immunological and molecular biological procedures. PCR data showed that MRP1 was more highly expressed when compared to another ATP-binding cassette family, multi-drug resistant protein and the expression levels of MRP1 on the cells were specifically dose- and time-dependently depleted by the addition of SAL. These results were also evaluated by immunological procedures using FACS and western-blotting. Small interfering RNA coding a part of MRP1 was transfected to Raji cells to knock down the protein, and cell death was increased by 10% when vincristine was administered at a concentration as low as 10 ng/mL compared to non-transfected cells. These results indicated that SAL possesses the potential to enhance the anticancer activites of low-concentrations of vincristine by the down-regulating the MRP1 gene expression to inhibit the multidrug resistance by binding to the target ligand Gb3-glycosphingolipid on Burkitt's lymphoma cells.

Antiproliferative effects of galectin-1 from Rana catesbeiana eggs on human leukemia cells and its binding proteins in human cells.

Galectin-1 from American bullfrog, RCG1, was isolated to high purity, and its growth inhibitory properties against human cells were examined. The results demonstrated that highly purified RCG1 induced large cell aggregates and revealed cell-type-specific growth inhibition. It significantly inhibited all human leukemia cell lines tested such as HL-60, U937, and K562 cells but did not inhibit human colon cancer cell line, Colo 201, or mouse mammary tumor cell line FM3A cells. Although most of the galectin-induced growth inhibitions are known to be apoptic, RCG1 induced growth arrest and neither apoptosis nor necrosis. RCG1-mediated growth inhibition was specifically suppressed by the corresponding sugar, lactose, but not by sucrose or even the structurally similar sugar, melibiose. Several studies have reported that galectin-mediated biological functions were modulated by charge modification. Since the high purity of RCG1 was demonstrated but a moderate degree of growth inhibition occurred, it is possible protein charge modification was examined by isoelectric focusing, and it was found to be highly heterogeneous in charge. RCG1 binding proteins in human cells were analyzed by lectin blotting using biotinylated RCG1, and lectin blotting revealed that in human cell extracts the specific proteins at molecular weight 37 and 50 kDa possessed the responsive features of RCG1 binding and lactose competition.

Cytotoxicity and glycan-binding profile of a D-galactose-binding lectin from the eggs of a Japanese sea hare (Aplysia kurodai).

A divalent cation-independent 16 kDa D-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized D-galactose and D-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl D-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galß1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k(ass) and k(diss) values are 2.4 × 10³ M⁻¹ s⁻¹ and 3.8 × 10⁻³ s⁻¹, respectively. AKL-2 appeared cytotoxicity against both Burkitt's lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.

Application of the adductome approach to assess intertissue DNA damage variations in human lung and esophagus.

Methods for determining the differential susceptibility of human organs to DNA damage have not yet been explored to any large extent due to technical constraints. The development of comprehensive analytical approaches by which to detect intertissue variations in DNA damage susceptibility may advance our understanding of the roles of DNA adducts in cancer etiology and as exposure biomarkers at least. A strategy designed for the detection and comparison of multiple DNA adducts from different tissue samples was applied to assess esophageal and peripherally- and centrally-located lung tissue DNA obtained from the same person. This adductome approach utilized LC/ESI-MS/MS analysis methods designed to detect the neutral loss of 2'-deoxyribose from positively ionized 2'-deoxynucleoside adducts transmitting the [M+H](+)>[M+H-116](+) transition over 374 transitions. In the final analyses, adductome maps were produced which facilitated the visualization of putative DNA adducts and their relative levels of occurrence and allowed for comprehensive comparisons between samples, including a calf thymus DNA negative control. The largest putative adducts were distributed similarly across the samples, however, differences in the relative amounts of putative adducts in lung and esophagus tissue were also revealed. The largest-occurring lung tissue DNA putative adducts were 90% similar (n=50), while putative adducts in esophagus tissue DNA were shown to be 80 and 84% similar to central and peripheral lung tissue DNA respectively. Seven DNA adducts, N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG), 1,N(6)-etheno-2'-deoxyadenosine (varepsilondA), alpha-S- and alpha-R-methyl-gamma-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-PdG(1), 1,N(2)-PdG(2)), 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxy-pyrimido[1,2-a]purine-(3H)-one (8-OH-PdG) and the two stereoisomers of 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (6-OH-PdG) were unambiguously detected in all tissue DNA samples by comparison to authentic adduct standards and stable isotope dilution and their identities were matched to putative adducts detected in the adductome maps.

Increased formation of hepatic N2-ethylidene-2'-deoxyguanosine DNA adducts in aldehyde dehydrogenase 2-knockout mice treated with ethanol.

N2-ethylidene-2'-deoxyguanosine (N2-ethylidene-dG) is a major DNA adduct induced by acetaldehyde. Although it is unstable in the nucleoside form, it is relatively stable when present in DNA. In this study, we analyzed three acetaldehyde-derived DNA adducts, N2-ethylidene-dG, N2-ethyl-2'-deoxyguanosine (N2-Et-dG) and alpha-methyl-gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (alpha-Me-gamma-OH-PdG) in the liver DNA of aldehyde dehydrogenase (Aldh)-2-knockout mice to determine the influence of alcohol consumption and the Aldh2 genotype on the levels of DNA damage. In control Aldh2+/+ mice, the level of N2-ethylidene-dG adduct in liver DNA was 1.9 +/- 0.7 adducts per 10(7) bases and was not significantly different than that of Aldh2+/- and -/- mice. In alcohol-fed mice (20% ethanol for 5 weeks), the adduct levels of Aldh2+/+, +/- and -/- mice were 7.9 +/- 1.8, 23.3 +/- 4.0 and 79.9 +/- 14.2 adducts per 10(7) bases, respectively, and indicated that adduct level was alcohol and Aldh2 genotype dependent. In contrast, an alcohol- or Aldh2 genotype-dependent increase was not observed for alpha-Me-gamma-OH-PdG, and N2-Et-dG was not detected in any of the analyzed samples. In conclusion, the risk of formation of N2-ethylidene-dG in model animal liver in vivo is significantly higher in the Aldh2-deficient population and these results may contribute to our understanding of in vivo adduct formation in humans.

Increased DNA damage in ALDH2-deficient alcoholics.

Drinking alcohol is a risk factor for cancers of the oral cavity, pharynx, larynx, and esophagus. Although many studies suggest that acetaldehyde, a major metabolite of orally ingested alcohol, plays a crucial role in cancer initiation, the link between the aldehyde dehydrogenase-2 (ALDH2) genotype and acetaldehyde-derived DNA damage has not yet been explored. We have developed a sensitive and quantitative method for detecting the acetaldehyde-derived DNA adducts, N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG), alpha-S- and alpha-R-methyl-gamma-hydroxy-1,N(2)-propano-2'-deoxyguanosine (alpha-S-Me-gamma-OH-PdG and alpha-R-Me-gamma-OH-PdG), and N(2)-(2,6-dimethyl-1,3-dioxan-4-yl)-deoxyguanosine (N(2)-Dio-dG), by using liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) and stable-isotope internal standards. We determined the DNA adducts in 44 blood DNA samples from Japanese alcoholic patients. The levels of three acetaldehyde-derived DNA adducts, N(2)-Et-dG, alpha-S-Me-gamma-OH-PdG, and alpha-R-Me-gamma-OH-PdG, were significantly higher in alcoholics with the ALDH2 1/2 2 genotype compared to those with the ALDH2 1/2 1 genotype. N(2)-Dio-dG was not detected in any of the DNA samples analyzed. These results provide molecular evidence that the ALDH2 genotype affects the genotoxic damage caused by acetaldehyde.

Development of the adductome approach to detect DNA damage in humans.

The development of new strategies designed to detect DNA damage caused by oxidative stress and other means may advance our understanding of the roles of such types of damage in the etiology of cancers, in aging processes, and as biomarkers of exposure. A DNA adduct detection method that uses liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) to detect multiple DNA adducts in human lung tissue is reported herein. This adductome analysis strategy is designed to detect the neutral loss of 2 -deoxyribose from positively ionized 2 -deoxynucleoside adducts in multiple reaction ion monitoring mode (MRM) transmitting the [M + H](+) > [M + H - 116](+) transition over a total of 374 transitions in the mass range from m/z 228.8 to m/z 602.8. Data analysis is optimized and coupled with a comprehensive manual screening process designed to minimize the number of artifactual adducts appearing in the final analysis. In the final analysis, putative adducts were organized into an adductome map and unambiguous confirmation of selected oxidative adducts were made by stable isotope dilution and comparison to authentic standards. The future applications of this method are discussed.