Construction of an infectious plasmid clone of Muscovy duck parvovirus by TA cloning and creation of a partially attenuated strain.

Muscovy duck parvovirus (MDPV) infection is a highly contagious and fatal disease of Muscovy ducklings. The infectious clone methodology is a valuable tool to study the pathogenic mechanisms of viruses, but no infectious clone of MDPV is yet available. In this study, a plasmid clone containing the full-length genome of MDPV was constructed using the TA cloning methodology. This MDPV clone was found to be infectious after transfection of primary Muscovy duck embryo fibroblast cells and passage in embryonated Muscovy duck eggs. Site-directed mutagenesis showed that the K75N mutation in the VP1 protein of MDPV resulted in the partial attenuation of the virus. The availability of an MDPV infectious clone can facilitate investigation of the pathogenic mechanisms of MDPV and development of vaccines against diseases caused by MDPV.

Identification and functional analysis of the cytolethal distending toxin gene from Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Cytolethal distending toxins (CDTs) are a family of protein cytotoxins that cause cell cycle arrest and apoptosis in eukaryotic cells. Whole-genome sequencing analysis showed that Av. paragallinarum contains cdtABC genes. Filter-sterilized lysates prepared from Av. paragallinarum or from recombinant Escherichia coli expressing cdtABC genes exhibited CDT activity on HeLa cells and chicken embryo fibroblast (DF-1) cells. In vitro DNase assays showed that purified recombinant CdtB has DNase activity. Polymerase chain reaction and sequencing analysis revealed that the cdtABC genes are present in all strains of Av. paragallinarum examined in this study. This is the first report of the identification and functional analysis of cdtABC genes from Av. paragallinarum. The gene products of cdtABC genes may be involved in the pathogenesis of the disease caused by Av. paragallinarum.

Gas chromatography-mass spectrometry determination of conjugated linoleic acids and cholesterol oxides and their stability in a model system.

A gas chromatography-mass spectrometry (GC-MS) method was developed to simultaneously separate cholesterol, eight cholesterol oxidation products (COPs), and two conjugated linoleic acids (9-cis,11-trans-CLA and 10-trans,12-cis-CLA) and to evaluate their stability in a model system during heating. Among four capillary columns tested, an Equity-5 column with low-polar stationary phase provided better resolution within 30 min. A high-performance liquid chromatography method was also developed to determine cholesterol hydroperoxides by using a YMC C30 column with diphenyl-1-pyrenylphosphine as fluorescence reagent. No formation of COPs or degradation of cholesterol and CLAs occurred at 100 degrees C, but the levels of COPs rose drastically at 150 degrees C. The first-order rate of cholesterol degradation declined following a rise in CLA concentration. For 0-, 100-, and 500-microg/ml CLA levels, the formation profiles of 7-hydroxycholesterol, 7-ketocholesterol, and 5,6-epoxycholesterol at 150 degrees C were fitted as multiple first-order curves, whereas a single first-order model could adequately describe 7-hydroperoxycholesterol and cholestane-3beta,5alpha,6beta-triol formation. A CLA-to-cholesterol mole ratio of 0.49 was required to prevent cholesterol oxidation at 150 degrees C.

Processes that affect electrospray ionization-mass spectrometry of nucleobases and nucleosides.

Due to the complexity of electrospray ionization processes and the many factors that affect the ion signal, optimization of electrospray ionization methods to gain ultimate sensitivity for analysis of nucleobases and nucleosides may not be straightforward. In this work, we investigated the effect of the pK a and the gas-phase basicity of analyte and other electrolytes on the [M+H](+) ion signal for 11 select nucleobases and nucleosides in 50% methanol:water solution. Solution chemistry plays a role in the electrospray signal for all analytes, but gas-phase chemistry may be important for compounds with pK a <3 depending on the solution composition. For compounds with pK a <3, gas-phase proton transfer reactions can be promoted to increase analyte electrospray response by the addition of ammonium acetate to the solution.

Analysis of phytochelatin-cadmium complexes from plant tissue culture using nano-electrospray ionization tandem mass spectrometry and capillary liquid chromatography/electrospray ionization tandem mass spectrometry.

Phytochelatins (PCs, also known as class III metallothioneins), a family of sulfhydryl-rich peptides with the formula (gamma-GluCys)(n)Gly(Pc(n), n = 2-11), are induced in plants, yeast and fungi exposed to heavy metals, and are thought to detoxify metals by forming PC- metal complexes. Although PCs have been detected, PC- metal complexes have not been well characterized. In this work, nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and capillary liquid chromatography/electrospray ionization tandem mass spectrometry (capillary LC/ESI-MS/MS) methods were used to analyze PC - Cd complexes isolated from Datura innoxia, also known as Jimsonweed, cell culture exposed to Cd. With nano-ESI-MS/MS and capillary LC/ESI-MS/MS we could simultaneously detect the presence of PCs and PC - Cd complexes from plant cell extracts, unambiguously identify these species and elucidate the nature of individual PC - Cd complexes. Phytochelatins with n = 3-6 were detected, as were PC - Cd complexes with PC(3), PC(4) and PC(5). This is the first study to report the size and nature of native PC - Cd complexes from plant tissue samples. These results demonstrate that the direct analysis of plant extracts using nano-ESI-MS/MS and capillary LC/ESI-MS/MS methods is simple and sensitive to the range of PCs and PC - Cd complexes in plants. Hence these methods open up new opportunities for further quantitative analysis of PCs and PC - metal complexes in cell culture and plant systems to understand the relationship between the biosynthesis of these compounds and metal tolerance.

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

Quantitative analysis of the DNA adduct N2,3-ethenoguanine using liquid chromatography/electrospray ionization mass spectrometry.

The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (epsilon Gua), a promutagenic DNA adduct of vinyl chloride exposure. The response of epsilon Gua to isotopically labelled internal standard [13C4]epsilon Gua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol microliter-1. We obtained an accuracy of 86 +/- 14% by analyzing chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic epsilon Gua. The analysis of CEO-treated calf thymus DNA samples not enriched with authentic epsilon Gua provided a precision of 15%. The detection limits with a signal-to-noise ratio (S/N) 2.5:1 were obtained in the determination of authentic epsilon Gua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol epsilon Gua with S/N = 3:1. The applicability of the method was established by determining epsilon Gua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of epsilon Gua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, epsilon Gua was quantitated at 0.06 +/- 0.01 pmol mg-1 DNA.

A video-based system for acquiring biomechanical data synchronized with arbitrary events and activities.

A video-based data acquisition and interactive multimedia data extraction system are described for measuring and synchronizing large quantities of biomechanical analog data with arbitrary events and activities. Analog signals from up to 32 channels are digitized, frequency-shift key (FSK) coded, and recorded directly onto the audio tracks of a video tape in synchronization with the video information. The data acquisition system includes an A/D converter that digitizes up to 16 multiplexed channels of 8-b data at a fixed sample rate between 60 and 960 Hz, and an FSK modem that transfers the data onto one of two VHS high fidelity (20 Hz-20 kHz bandwidth) audio tracks. Twenty megabytes of digitized data and time codes, along with associated video and normal audio are contained on a conventional 120-min video tape. An analyst interactively reviews the video tape off-line using a computer-controlled VCR and identifies specific events that divide arbitrary activities into time segments. The computer automatically extracts the biomechanical data corresponding to each time segment for further processing or analysis. This system is useful for ergonomics, gait analysis, sports medicine, sleep laboratory, biomechanics, or any application where complex visual events are synchronized with low-frequency analog data.

1,3-butadiene: cancer, mutations, and adducts. Part V: Hemoglobin adducts as biomarkers of 1,3-butadiene exposure and metabolism.

1,3-Butadiene (BD) is an important chemical used largely in the manufacture of synthetic rubber and thermoplastic resins. In addition, it has been identified in cigarette smoke, automobile exhaust, and gasoline vapor. The objective of this research was to develop highly sensitive and specific assays for the detection and quantitation of hemoglobin adducts of three BD metabolites: 1,2-epoxy-3-butene (BDO), 1,2,3,4-diepoxybutane (BDO2), and 1,2-dihydroxy-3,4-epoxybutane (BDO-diol). We have successfully developed an assay for both N-(2-hydroxy-3-butenyl)valine (HBVal) and N-(2,3,4-trihydroxybutyl)valine (THBVal) in hemoglobin. The six adducts measured were the two diastereomers (isomers I and II) of HBVal and the four diastereomers of THBVal (isomers I through IV, which were eluted as three peaks, 1, 2, and 3). HBVal and THBVal were measured in control and exposed B6C3F1 mice and Sprague-Dawley rats (1,000 ppm BD for 13 weeks at 6 hours/day, 5 days/week). In a second set of animal exposures, total THBVal was determined in B6C3F1 female mice (n = 5) exposed to 1,250 ppm BD for 1, 5, or 10 days (6 hours/day, 5 days/week). THBVal adducts were also monitored in occupationally exposed Chinese workers and nonoccupationally exposed U.S. laboratory workers. This study utilized the modified Edman degradation method of Törnqvist and colleagues (1986). Briefly, the samples were subjected to Edman degradation, Centricon-30 ultrafiltration, washing on C18 columns, and acetylation for isomers of THBVal only, followed by gas chromatography-mass spectrometry (GC-MS) quantitation. For the HBVal assay, an authentic internal standard globin alkylated with [2H6]BDO was used; for the THBVal assay, a synthesized external standard, THB[13C5]Val, was used after Edman degradation. The mean +/- SD amounts of total HBVal measured in exposed mice (in pmol/g globin) were 16,560 +/- 3,910 for female mice (n = 4) and 12,400 +/- 2,030 for male mice (n = 5). The corresponding values for rats were 8,690 +/- 930 for female rats (n = 5) and 5,480 +/- 2,880 for male rats (n = 3). The total amount of THBVal (eluted peaks 1, 2, and 3) in male mice (n = 5) was 78,900 +/- 13,700; and in females (n = 2) was 56,100 +/- 100. In male rats (n = 3), the detected value was 9,650 +/- 1,620 and in females (n = 3) the value was 21,600 +/- 6,780. In control male mice (n = 4), the total level of THBVal isomers was approximately 27 pmol/g globin. In a control male rat, total THBVal was approximately 15 pmol/g globin. In the time course study, the amount of THBVal adducts increased linearly with exposure, resulting in values of 4,200 +/- 830, 19,760 +/- 1,780, and 35,940 +/- 3,460 pmol/g globin following 1, 5, or 10 days of exposure to 1,250 ppm BD, respectively. Detection of HBVal in human samples was difficult due to low concentrations of adducts and a high background in the chromatograms. In a pooled sample from 4 individuals, we performed multiple separations with high-pressure liquid chromatography (HPLC) of the derivatized adducts and detected 4.6 pmol/g globin (that is, 2.7 and 1.9 pmol/g globin for isomers I and II, respectively). We measured the amounts of THBVal in both nonoccupationally exposed U.S. laboratory workers and occupationally exposed workers from a polybutadiene plant in China. The mean total amount of THBVal among the U.S. laboratory workers was 36 +/- 23 pmol/g globin for nonsmokers (n = 7) and 40 +/- 9 for smokers (n = 4), compared with a mean total amount of 39 +/- 13 pmol/g globin in a control set of Chinese workers (n = 25). These control values are overestimations of the true values because the amounts of THBVal in globin samples from other unexposed individuals (15 of 51) were below our limit of detection. BD-exposed Chinese workers had a total amount of 88 +/- 59 pmol/g globin THBVal. The difference between smokers and nonsmokers was not significant, whereas the difference between control and exposed Chinese workers was highly significant (p < 0.001).

Comparison between using spectral analysis of electrogoniometer data and observational analysis to quantify repetitive motion and ergonomic changes in cyclical industrial work.

Spectral analysis of continuously measured joint angles using an electrogoniometer was considered as a potentially efficient method for quantifying exposure to physical stress in repetitive manual work. The method was previously demonstrated in the laboratory but has not yet been tested extensively in the field. Spectral analysis was compared against observational analysis, consisting of time-and-motion study and posture classification. Six industrial jobs were selected: (1) press operation, (2) large parts hanging, (3) product packaging, (4) small parts hanging, (5) parts counting and sorting and (6) construction vehicle operation. The posture angle data were synchronized with activities on the video using an interactive multimedia video data acquisition system. Motion for every joint was analyzed using both spectral analysis and observational analysis. Joint angles for the wrist, elbow and shoulder were directly measured using electrogoniometers. Visual posture classification involved determining joint angles from a frozen videotape image sampled three times per s. Repetitiveness was quantified for observational analysis using time study to measure the frequency that specific motions repeat, while spectral analysis measured repetitiveness as the frequency where spectral peaks occurred. Spectral analysis agreed closely with observational analysis. Correlation between the repetition frequencies obtained using time study and spectral analysis was 0.97, with no statistically significant difference observed. Average sustained posture was quantified as the mean, and posture deviation as the RMS angle of joint motion. No statistically significant differences between data obtained using posture classification or spectral analysis were observed for either posture deviation or sustained posture. Since posture classification was very limited in resolution and often contained measurement errors caused by poor joint visibility, the correlation between the postural classification and spectral analysis was 0.77 for sustained posture and 0.53 for posture deviation. When considering only large motions that exceeded the posture classification angle precision, the correlation between postural classification and spectral analysis was 0.81 for sustained posture and 0.81 for posture deviation. Spectral analysis of electrogoniometer data were, therefore, an efficient method for analyzing repetitive manual work that obtained equivalent results, and was more precise than observational analysis.

Exposure assessment of biomechanical stress in repetitive manual work using frequency-weighted filters.

A quantitative exposure assessment strategy for physical stress associated with repetitive manual tasks is proposed using continuous biomechanical data measured directly from electrogoniometers or force sensors. This paper describes an efficient method for reducing large quantities of biomechanical data into a quantifiable metric that accounts for recognized musculoskeletal exposure factors, including repetitiveness, postural or forceful exertion stress, and duration. A frequency domain approach is used for averaging elemental data recorded for repetitive cycles. Parameters for frequency-weighted filters are developed using psychophysical data for equivalent discomfort levels resulting from repetitive movements of different amplitudes and frequencies. These filters enable continuous biomechanical data to be filtered and integrated, resulting in a single quantity corresponding to psychophysical response characteristics for repetitive motion stress. It is anticipated that a similar approach may be used for epidemiological response characteristics. Applications of this theory may make it possible for assessing exposure to physical stress in a manner analogous to the way in which sound level meters are used for measuring exposure to acoustic noise. Repetitive wrist flexion and localized discomfort was used for demonstrating the feasibility of this approach. Suitable data reduction techniques are necessary for evaluating work methods, job designs, and for conducting large scale detailed epidemiological investigations of cumulative trauma disorder risk factors. Frequency-weighted filters based on human response to physical stress at different frequencies can greatly simplify exposure analysis and ultimately may make it possible for quantitative exposure limits to be established.

Synthesis, characterization, and in vitro quantitation of N-7-guanine adducts of diepoxybutane.

Diepoxybutane (DEB) is an important metabolite of 1,3-butadiene (BD), a high-volume industrial chemical classified as a probable human carcinogen. Rodent inhalation studies show strikingly high sensitivity of mice to carcinogenic effects of butadiene compared to rats, which has been linked to differences in metabolism. Both species convert BD to 3,4-epoxy-1-butene (EB), but mice further oxidize a significantly greater part of EB to DEB. DEB is a potent bifunctional genotoxic agent which is 100-fold more mutagenic than EB and is likely to be involved in BD-induced carcinogenesis. Identification of specific BD-induced DNA adducts is critical to understanding the mechanism of its biological activity. We have previously described reactions of EB with guanine and adenine as nucleobases, nucleosides, and constituents of DNA. In this work, DEB-induced guanine adducts were isolated and structurally characterized by UV spectroscopy, mass spectrometry, and nuclear magnetic resonance. When guanosine was reacted with DEB in glacial acetic acid followed by hydrolysis in hydrochloric acid, three products were isolated: N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (DEB-Gua I, major adduct), N-7-(2',4'-dihydroxy-3'-chlorobut-1'-yl)guanine (DEB-Gua II), and N-7-(2',3'-dihydroxy-4'-acetoxybut-1'-yl)guanine (DEB-Gua III). We suggest initial formation of the N-7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine intermediate followed by nucleophilic substitution at the 3',4'-epoxy ring with hydroxide, chloride, or acetate anions to give DEB-Gua I, II, or III, respectively. DEB-Gua I and the epoxy intermediate were also isolated from hydrolysates of DEB-exposed calf thymus DNA (CT DNA). N-7-Guanine adducts are known to undergo spontaneous and enzymatic depurination producing apurinic sites. If not repaired before DNA replication, apurinic sites can give rise to mutations and ultimately cancer. The extent of alkylation at the N-7 of guanine in DEB-exposed DNA (58.7 +/- 1.1 adducts/10(3) normal guanines) was similar to that previously reported for CT DNA exposed to EB at the same molar ratio. Since EB and DEB appear to induce comparable levels of overall DNA alkylation at the conditions applied in this work, other factors, such as formation of DNA cross-links by DEB but not EB or differences in repair of EB and DEB adducts, may be responsible for the differences in mutagenicity.

Evaluation of iliopsoas compartment disorders by computed tomography.

BACKGROUND: Iliopsoas compartment lesion frequently goes undiagnosed at clinical presentation. Therefore, even delayed diagnosis may depend on radiographic techniques. Computed tomography (CT) can play an important role in the identification and localization of these lesions. This report concerns the experience of using CT to evaluate the iliopsoas compartment disorder. METHODS: Retrospective review of CT scans for 42 patients who had abnormalities of the iliopsoas compartment revealed that inflammation was responsible in 20, hemorrhage in 8, and tumor in 14 patients. Diagnoses were proven by surgery or biopsy/aspiration in all cases except in five cases whose CT findings correlated with their clinical pictures. A comparison was made among the surgical data, the CT and plain abdominal films. In addition, a comparative evaluation of CT features was also made. RESULTS: By using a hypodense area for the diagnosis of psoas abscess, the sensitivity, specificity and accuracy of CT were 100%, 77%, and 88%, respectively. By using a diffuse involvement of the entire muscle for the diagnosis of hemorrhage, the sensitivity, specificity and accuracy of CT were 63%, 62%, and 62% respectively. By using a bone destruction for the diagnosis of tumors, the sensitivity, specificity and accuracy of CT were 79%, 39%, and 69%, respectively. Evaluation of the iliopsoas compartment lesions was 91% sensitive by CT and 40% sensitive by plain X-ray film. CONCLUSIONS: It is difficult to differentiate iliopsoas abscesses, hematomas and neoplasms without knowing the clinical history. However, when coupled with percutaneous aspiration biopsy, CT has proved to be an effective, rapid means to diagnose and guide the therapy of psoas lesions.

13-cis retinoic acid enhances in vivo B-lymphocyte differentiation in patients with common variable immunodeficiency.

Retinoic acid (RA) has been demonstrated to drive both phenotypic and functional in vitro differentiation of B cell hybridomas from patients with common variable immunodeficiency (CVI) who manifest an "intrinsic" defect in terminal B cell differentiation (J Exp Med 1988;168: 55-71). Therefore, we conducted an open trial to determine the effects of oral 13-cis RA (0.5 mg/kg/day; 12 weeks receiving and 12 weeks without drug) on in vivo B cell differentiation in subjects with CVI. At various times before, during, and after drug administration, patients' B cells were tested for changes in cell-surface phenotype and in vitro immunoglobulin production in response to recombinant cytokines. Before 13-cis RA, all patients had decreased Leu-8 coexpression on CD20+ cells. Seven of eight subjects demonstrated "normalization" of this phenotype after 8 to 16 weeks of 13-cis RA administration. Patients whose B cells demonstrated more than normal CD20 display also had a fall toward normal in this parameter. These effects persisted for 6 to 12 weeks after drug was stopped. It appears that 13-cis RA drives B cells of patients with CVI to express a more differentiated cell-surface phenotype and may promote functional differentiation in some patients.

Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA.

1,3-Butadiene (BD) is a high-volume industrial chemical and a common environmental pollutant. Although BD is classified as a "probable human carcinogen", only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of BD compared to rats (approximately 10(3)-fold), making interspecies extrapolations difficult. Identification and quantitation of specific BD-induced DNA adducts are important for improving our understanding of the mechanisms of BD biological effects and for explaining the observed species differences. Covalent binding of BD to DNA is probably due to its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Both EB and DEB are direct mutagens producing frameshift and point mutations at both A:T and G:C base pairs. DEB is 100 times more mutagenic than EB and is found in quantity only in tissues of the most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to BD could be due to greater exposure to DEB. The present work was initiated in order to isolate and structurally characterize DEB-induced adenine adducts. The adducts were formed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine. These adducts underwent acid-catalyzed hydrolysis to their corresponding (2',3',4'-trihydroxybut-1'-yl)adenines upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-DEB-Ade). N-3-DEB-Ade and N6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3-DEB-Ade (13/10(3) normal Ade) and N6-DEB-Ade (5/10(3) normal Ade) were slightly lower than those of the corresponding EB-induced adducts in similar experiments, suggesting comparable reactivity of the two epoxy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of BD-induced adenyl DNA adducts in vitro and in vivo.

High-performance liquid chromatographic separation of the biotransformation products of oxaliplatin.

A novel single reversed-phase HPLC system was developed for separating oxaliplatin and its biotransformation products formed in rat plasma. The major stable biotransformation products of oxaliplatin formed in rat plasma were identified as Pt(dach)(Cys)2, Pt(dach)(Met) and free dach. The minor biotransformation products Pt(dach)Cl2, Pt(dach)(GSH) and Pt(dach)(GSH)2 could also be resolved from other Pt-dach complexes. Among these biotransformation products, the identification of Pt(dach)(Met) was further confirmed by LC-ESI-MS, and the identification of Pt(dach)(Cys)2, Pt(dach)(GSH), Pt(dach)(GSH)2 and free dach was confirmed by atomic absorption and double isotope labeling. This HPLC technique should prove useful for separating and identifying the biotransformation products of Pt-dach drugs such as oxaliplatin, ormaplatin and Pt(dach)(mal) in biological fluids. This will allow a more complete characterization of the pharmacokinetics and biotransformations of these Pt-dach drugs, which should in turn lead to a better understanding of the mechanisms leading to their toxicity and efficacy.

Detection of 1,N6-ethenoadenine in rat urine after chloroethylene oxide exposure.

The four etheno adducts of vinyl chloride formed in DNA, 1,N6-ethenoadenine (epsilonA), 3,N4-ethenocytosine, 1,N2-ethenoguanine and N2,3-ethenoguanine were previously reported to be released from DNA by a family of enzymes in the base-excision repair pathway (Dosanjh et al., Proc. Natl Acad. Sci. USA, 91, 1024-1028, 1994; Hang et al., Carcinogenesis, 17, 155-157, 1996; Hang et al., Proc. Natl Acad. Sci. USA, 94, 12869-12874, 1997). Adducts excised from DNA by glycosylases are usually excreted in urine and have been reported to be potential biomarkers of DNA damage in exposed individuals. In this study, we report the detection of epsilonA in the urine of rats exposed to chloroethylene oxide (CEO) using immunoaffinity columns made with specific monoclonal antibodies for enrichment, followed by quantitation by HPLC with fluorescence detection. Chemical analysis of urine samples revealed the presence of a compound chromatographically identical to authentic epsilonA standard. This compound was confirmed by mass spectral analysis. EpsilonA was present in urine of control and CEO-treated rats, with the latter having up to 50-fold greater amounts. The cumulative excretion of epsilonA reached a plateau between 24 and 48 h post-exposure. While it is clear that CEO treatment results in increased excretion of epsilonA, the exact source of the adduct is unknown. When rats were administered epsilonA i.v., approximately 10% of the administered dose was excreted in urine. This research demonstrates that urinary excretion of epsilonA may be a potential biomarker for in vivo alkylation of DNA and nucleotide pools.

Quantitation of 1,N6-ethenoadenine in rat urine by immunoaffinity extraction combined with liquid chromatography/electrospray ionization mass spectrometry.

A fast, highly specific analytical method was developed to quantify 1,N6-ethenoadenine (epsilonA) in urine of rats. epsilonA is a highly mutagenic DNA adduct generated by vinyl chloride (VC) exposures as well as endogenously from lipid peroxidation. epsilonA was concentrated through extraction from rat urine by immunoaffinity chromatography and quantitated by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The average epsilonA recovery by immunoaffinity extraction was 66%. The LC/ESI-MS selected-ion monitoring (SIM) of the response ratio of epsilonA to its isotopically labeled internal standard [15N5]epsilonA was linear (r2 = 0.999) and reproducible from 0.15 to 30 pmol/injection. The detection limit obtained in the routine analysis of urine of unexposed rats was 270 fmol/sample with a signal-to-noise ratio (S/N) 3:1. The concentration of endogenous epsilonA was determined to be 21.6 +/- 14.8 pmol/mL (3 rats). Following portal injection of chloroethylene oxide (CEO; the putative active metabolite of VC), the rate of epsilonA excretion in urine was greatest from 0 to 24 h, with approximately 90% of the CEO-induced epsilonA excreted. By 132 h, the excretion of epsilonA was similar to pretreatment amounts. The accuracy of the quantitation was 107 +/- 6% (n = 4), established by analyzing urine of an unexposed rat spiked with authentic epsilonA. These data indicate that the LC/ESI-MS with immunoaffinity extraction method is precise and accurate for epsilonA quantification. The measurement of epsilonA in urine provides a potential biomarker for exposure to chemicals and processes that form this adduct.

Unique disulfide bond structures found in ST8Sia IV polysialyltransferase are required for its activity.

NCAM polysialylation plays a critical role in neuronal development and regeneration. Polysialylation of the neural cell adhesion molecule (NCAM) is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which contain sialylmotifs L and S conserved in all members of the sialyltransferases. The members of the ST8Sia gene family, including ST8Sia II and ST8Sia IV are unique in having three cysteines in sialylmotif L, one cysteine in sialylmotif S, and one cysteine at the COOH terminus. However, structural information, including how disulfide bonds are formed, has not been determined for any of the sialyltransferases. To obtain insight into the structure/function of ST8Sia IV, we expressed human ST8Sia IV in insect cells, Trichoplusia ni, and found that the enzyme produced in the insect cells catalyzes NCAM polysialylation, although it cannot polysialylate itself ("autopolysialylation"). We also found that ST8Sia IV does not form a dimer through disulfide bonds. By using the same enzyme preparation and performing mass spectrometric analysis, we found that the first cysteine in sialylmotif L and the cysteine in sialylmotif S form a disulfide bridge, whereas the second cysteine in sialylmotif L and the cysteine at the COOH terminus form a second disulfide bridge. Site-directed mutagenesis demonstrated that mutation at cysteine residues involved in the disulfide bridges completely inactivated the enzyme. Moreover, changes in the position of the COOH-terminal cysteine abolished its activity. By contrast, the addition of green fluorescence protein at the COOH terminus of ST8Sia IV did not render the enzyme inactive. These results combined indicate that the sterical structure formed by intramolecular disulfide bonds, which bring the sialylmotifs and the COOH terminus within close proximity, is critical for the catalytic activity of ST8Sia IV.

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry method for analysis of endogenous and exogenous N7-(2-hydroxyethyl)guanine in rodents and its potential for human biological monitoring.

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS) method was developed for quantitating N7-(2-hydroxyethyl)guanine (N7-HEG) with excellent sensitivity and specificity. [4,5,6,8-(13)C(4)]-N7-HEG was synthesized, characterized, and quantitated using HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) so it could serve as an internal standard. After being converted to its corresponding xanthine and derivatized with pentafluorobenzyl (PFB) bromide twice, the PFB derivative of N7-HEG was characterized using GC/EC/NCI-HRMS carried out at full scan mode. The most abundant fragment was at m/z 555, with a molecular formula of C(21)H(9)N(4)O(3)F(10), resulting from the loss of one PFB group. By monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard), this assay demonstrated a linear relationship over a range of 1 fmol to 1 pmol of N7-HEG versus 20 fmol of [(13)C(4)]-N7-HEG on column. The limit of detection (LOD) for the complete assay was 600 amol (S/N = 5) injected on column. The variation of this assay was within 15% from 1 to 20 fmol of N7-HEG versus 2 fmol of [(13)C(4)]-N7-HEG with four replications for each calibration standard. Two hundred to three hundred micrograms of spleen DNA of control rats and mice and 100 microg of spleen DNA of rats and mice exposed to 3000 ppm ethylene for 6 h/day for 5 days were analyzed using GC/EC/NCI-HRMS. The amounts of N7-HEG varied from 0.2 to 0.3 pmol/micromol of guanine in tissues of control rats. Ethylene-exposed animals had 5-15-fold higher N7-HEG levels than controls. This assay was able to quantitate N7-HEG in 25-30 microg of DNA from human lymphocytes with excellent specificity. This was due in part to human tissues having 10-15-fold higher amounts of endogenous N7-HEG than rodents. These results show that this GC/EC/NCI-HRMS method is highly sensitive and specific and can be used in biological monitoring and molecular dosimetry and molecular epidemiology studies.