Fluorometric measurement of yessotoxins in shellfish by high-pressure liquid chromatography

A rapid HPLC method with fluorescence detection of yessotoxin (YTX) and its two analogs (45-OHYTX and norYTX) in mussels and scallops is presented. A dienophile reagent, DMEQ-TAD, was used for fluorescence labeling. YTX was measured in the range 1-100 ng. The method confirmed the occurrence of YTX and 45-OHYTX for the first time in mussels from Chile and New Zealand.     

Principles of magnetic resonance angiography

A strongly fluorescing 7-hydroxycoumarin (umbelliferone, U) oxidized in dilute (10 mumol/L-O, 1 mol/L) aqueous solution with CIO- or CIO- + H2O2 (but not with H2O2 alone) produces a strong chemiluminescence (CL). Light emission kinetics depends on the pH of solution (4.0-10.5) and the reaction has a low activation energy Ea = 31 +/- 2 kJ/mol (285-310 K). The spectrum covers the fluorescence of umbelliferone (400-550 nm, lambda max 460 nm). No red emission typical of 1 delta g, 1 sigma g, 1 sigma+g (O2)2 is observed either in the umbelliferone+CIO- or the umbelliferone +CIO- + H2O2 solution. The possible mechanism of CL and concomitant degradative oxidation of umbelliferone is discussed.     

Light from Maillard reaction: photon counting, emission spectrum, photography and visual perception

Several authors have reported on high-sensitivity measurement of oxygen-dependent low-level chemiluminescence (CL) from Maillard reactions (MR), i.e. nonenzymatic amino-carbonyl reactions between reducing sugars and amino acids (also referred to as nonenzymatic browning). Here we report for the first time, that light from Maillard reactions can be seen by the human eye and also can be photographed. In parallel with visual perception and photography CL was monitored by means of a CL-detection programme of a liquid scintillation counter (LSC, single photon rate counting). CL emission spectrum was recorded by a monochromator-microchannel plate photomultiplier arrangement. CL intensity from reaction of 6-aminocaproic acid with D-ribose (200 mg each) in 5 mL H2O at pH 11 at 95 degrees C was high enough for visual perception after adaptation to absolute darkness. Reaction in dimethylsulphoxide (DMSO) exhibited strongly enhanced CL (10 mg each in 5 mL were sufficient for visual detection) and could be photographed (15 minutes' exposure, ASA 6400); all characteristics of Maillard specific CL (O2-dependence, no CL from nonreducing sugars, inhibition by sulphur compounds) remained. Visual detection of CL and measurement by LSC were in full concordance. The CL emission spectrum showed two broad peaks at around 500 nm and 695 nm. Fluorescence emission of the brown reaction mixture matched the blue-green part of the CL emission spectrum. Emission of visible light during Maillard reactions may partly originate from oxygen-dependent generation of excited states and energy transfer to simultaneously formed fluorescent products of the browning reaction.     

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.     

Minor products of reaction of DNA with alpha-acetoxytamoxifen

The drug tamoxifen shows evidence of genotoxicity and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen and these arise, at least in part, from its metabolite alpha-hydroxytamoxifen. This probably undergoes conjugation in the liver tissue to give an ester, which alkylates DNA. We have prepared alpha-acetoxytamoxifen as a model for this reactive intermediate and studied its reaction with DNA in vitro. The products of this reaction were chromatographically identical to DNA adducts found in the liver of rats treated with tamoxifen. We have isolated three of these products as the nucleosides TG1, TG2 and TA1 and identified them by ultraviolet, mass and proton magnetic resonance spectroscopy. TG1 and TG2 were tamoxifen-deoxyguanosine adducts in which the alpha-position of tamoxifen was linked to the amino group of guanine; TG1, (E)-4-[4-[2-(dimethylamino)ethoxy]phenyl]-3,4-diphenyl-2-(9beta-de oxyribofuranosyl-6-oxopurin-2-ylamino)-3-butene; TG2, (Z) isomer of TG1. In TG2, the tamoxifen group had undergone trans-cis isomerization. The minor product TA1 was a tamoxifen-deoxyadenosine adduct, where linkage was through the amino group of adenine: (E)-4-[4-[2-(dimethylamino) ethoxy]phenyl]-3,4-diphenyl-2-(9beta-deoxyribofuranosylpurin -6-ylamino)-3-butene. These three adducts accounted for >90% of the reaction products (approximately 67% TG1, 18% TG2 and 7% TA1); trace products included other stereoisomers of these and dinucleotide adducts which resisted enzymatic digestion.     

A continuous spectrophotometric method for the determination of monophenolase activity of tyrosinase using 3-methyl-2-benzothiazolinone hydrazone

A continuous spectrophotometric method for the rapid determination of monophenolase activity of tyrosinase is described. This method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone (MBTH) and the quinone products of the oxidation of various monophenols in the presence of tyrosinase. The chemical reaction between MBTH and o-quinone has been kinetically characterized, the lambda max and the molar absorptivity coefficients of the adducts have been calculated, and the stoichiometry of the reaction has been determined. The method is illustrated by measuring the enzymatic activity of mushroom tyrosinase during the hydroxylation of phenol and tyramine. The presence of MBTH in the reaction medium decreases the lag period present during the expression of monophenolase activity and the high epsilon values at 500-505 nm of the adducts make this method more sensitive than other continuous methods. The MBTH reaction in the presence of monophenols or o-diphenols has been optimized to stain tyrosinase obtained from different biological sources in electrophoresis gels.     

Levels and patterns of alcohol consumption using timeline follow-back, daily diaries and real-time "electronic interviews"

BACKGROUND: Basic knowledge of the substances involved in wound healing after photorefractive keratectomy (PRK) is essential for development of pharmacological intervention. We present preoperative and postoperative analysis of tear fluid extracellular matrix proteins and cytokines after PRK. METHODS: Tear fluid samples from 70 patients (72 eyes) who had PRK (38 women and 32 men, mean age 31.5 yr) were studied. Samples from 18 patients (18 eyes) were analyzed in two different studies. RESULTS: Mean preoperative tear fluid flow in the collection capillary (volume divided by tear collection time) varied from 4.5 to 22.5 microliters/min in five series of patients. It increased significantly during the first two postoperative days (range of means, 55.5 to 88.8 microliters/min, p < 0.01), and decreased to the preoperative level by day 7 (range of means, 9.7 to 18.2 microliters/min). The tenascin and cytokine release rates increased significantly during the first two days after PRK and returned to the preoperative level by day 7. Mean +/- standard error for tenascin: day 0 (5.2 +/- 1.9 ng/min); day 2 (22.7 +/- 6.1 ng/min; p = 0.02). Mean +/- standard error for HGF: day 0 (3.2 +/- 0.7 pg/min); day 1 (22.8 +/- 4.2 pg/min; p = 0.0003). Mean +/- standard error for TGF-beta 1: day 0 (63.3 +/- 19.6 pg/min); days 1-2 (826.2 +/- 253.7 pg/min; p = 0.001). Mean +/- standard error for VEGF: day 0 (166.0 +/- 29.6 pg/min); days 1-2 (824.4 +/- 165.1 pg/min; p = 0.0007). Mean +/- standard error for PDGF-BB: day 0 (0.42 +/- 0.19 pg/min); day 2 (27.6 +/- 5.8 pg/min; p = 0.0000). Mean +/- standard error for TNF-alpha: day 0 (9.5 +/- 2.6 pg/min); day 2 (28.6 +/- 5.9 pg/min; p = 0.003). Excluding PDGF-BB, all substances studied were present in normal human tear fluid. PDGF-BB was present in only 17% of the preoperative samples. CONCLUSION: Corneal wounding induces an increased release of several growth modulating cytokines which may be involved in healing processes.     

Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms

Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by 32P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B1, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected 32P-labeled DNA adducts produced by aflatoxin B1, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B1, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70 degrees C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.     

Liquid chromatography of aromatic amines with photochemical derivatization and tris(bipyridine)ruthenium(III) chemiluminescence detection

We have shown by flow injection that tris(bipyridyl)ruthenium(III) [Ru(bpy)3(3+)] chemiluminescence (CL) detection of some aromatic amines can be enhanced by on-line photochemical derivatization. Two of the aromatic amino acids, tryptophan, and tyrosine as well as the peptide phenylalanine-alanine and other primary aromatic amines such as L-dopa, phentermine, and tryptamine upon irradiation with UV light are found to give an increased CL signal on the order of 4-9 times that for nonirradiated compounds. For benzylamine, phenethylamine, and phenylalanine, the improved CL detectability upon photolysis is about 15-16 times better. Chemiluminescence detection limits of the photolyzed compounds are generally 2-20 pmol, significantly better than those by UV-Vis detection at 254 nm. GC-MS work has been done to identify the products of some of the photolysis reactions and explain the enhanced CL detectability. The fact that other aromatic amines without a one or two carbon spacer from the aromatic ring to the amine group such as aniline, m- and p-phenylenediamine, and N,N'-dimethylaniline did not show any CL signal improvement upon irradiation with UV light suggests that there is some selectivity in the reaction. CL detection of aromatic amino acids after on-line photochemical derivatization and HPLC has been shown.     

Plasma levels of parathyroid hormone, vitamin D, calcitonin, and calcium in association with endurance exercise

Nine male marathon runners were investigated during habitual training (week 0), after 3 weeks of training break (week 3), and after 2 weeks (week 5) and 4 weeks (week 7) of retraining. Maximal oxygen uptake, body fat (BF), and plasma levels of 25(OH)D3, 1,25(OH)2D3, parathyroid hormone (PTH), calcitonin (CT), albumin, and albumin-corrected calcium were determined throughout weeks 0-7. The maximal oxygen uptake decreased after training break and increased during retraining (P = 0.002). BF did not change significantly. Plasma 1,25(OH)2D3 was elevated after training break and decreased after 2 and 4 weeks of retraining [week 0: 44.0 +/- 3.7 (SEM) pg x 1(-1); week 3: 52.4 +/- 6.0 pg x 1(-1); week 5: 42.0 +/- 2.8 pg x 1(-1); week 7: 36.9 +/- 2.3 pg x 1(-1); P = 0.03]. Plasma 25(OH)D3 did not change significantly. Plasma PTH increased throughout the training break and retraining (week 0: 1.36 +/- 0.25 pmol x 1(-1); week 3: 2.02 +/- 0.43 pmol x 1(-1); week 5: 2.23 +/- 0.60 pmol x 1(-1); week 7: 2.63 +/- 0.34 pmol x 1(-1); P = 0.03). Albumin-corrected calcium values were transiently decreased during retraining (week 3: 2.77 +/- 0.08 mM; week 5: 2.47 +/- 0.05 mM; week 7: 2.66 +/- 0.07 mM; P = 0.01). Plasma CT did not change during training break, but was transiently decreased during retraining (week 0: 9.97 +/- 0.39 pmol x 1(-1); week 3: 9.91 +/- 0.37 pmol x 1(-1); week 5: 8.19 +/- 0.50 pmol x 1(-1); week 7: 9.02 +/- 0.45 pmol x 1(-1); P = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of homocysteine-thiolactone and alpha-methylhomocysteine-thiolactone with e-(aq) and OH-radical: a pulse radiolysis study

The efficient radiation protecting agents homocysteine-thiolactone x HCl (HCTL x HCl) and its alpha-alkylated derivative (alpha-methyl-homocysteine, alpha-MHCTL x HCl) have been investigated in respect to the identification of the primarily formed species after absorption of ionizing radiation using pulse radiolysis technique.The reaction of e-(aq) with the unprotonated form of HCTL (k = 2.1 x 10(9) dm3 mol(-1) s(-1)) is leading to the formation of a radical anion having two absorption bands: at 275 nm (epsilon = 2500 dm3 mol(-1) cm(-1)) and 510 nm (epsilon = 930 dm3 mol(-1) cm(-1)), which decay with 2k = 2.3 x 10(9) dm3 mol(-1) s(-1). The protonated form of HCTL reacts with e-(aq) with k = 4.0 x 10(10) dm3 mol(-1) s(-1). The OH-radicals react with HCTL with k = 1.95 x 10(9) dm 3 mol(-1) x s(-1) resulting in a transient spectrum with lambda(max) = 265 nm (epsilon =2000 dm3 mol(-1) cm(-1)). The transients disappear with 2k = 2.1 x 10(9) dm3 mol(-1) s(-1). The reactivity of e-(aq) with alpha-MHCTL was determined for both forms: for the protonated, k = 1.25 x 10(10) dm3 mol(-1) s(-1) and for the unprotonated, k = 2.6 x 10(9) dm3 mol(-1) s(-1). The transient absorption spectrum at pH = 8.4 shows two absorption bands: lambda = 275 nm (epsilon = 3500 dm3 x mol(-1) x cm(-1)) and 490 nm (epsilon = 1160 dm3 x mol(-1) x cm(-1)). The transients disappear with 2k = 2.2 x 10(9) dm3 x mol(-1) x s(-1). The reaction of OH with alpha-MHCTL x HCl,k = 8.4 x 10(9) dm3 x mol(-1) x s(-1) (pH = 8.6) is resulting in an absorption spectrum with lambda(max) < 260 nm and an absorption band at 350 nm (epsilon = 510 dm3 x mol(-1) x cm(-1)). Up to 50 micros after pulse the transients decay with 2k = 5.5 x 10(9) dm3 x mol(-1) x s(-1) and thereafter by a k = 8.4 x 10(9) dm3 x mol(-1) x s(-1) (pH = 8.6) is resulting in an absorption spectrum with lambda(max) < 260 nm and an absorption band at 350 nm (epsilon = 510 dm3 x mol(-1) x cm(-1)). Up to 50 micros after pulse the transients decay with 2k = 5.5 x 10(9) dm3 x mol(-1) x s(-1) and thereafter by a first order reaction. In addition, the formation of some products was also studied. The yield of ammonia resulting from alpha-MHCTL x HCl strongly depends on pH, e.g. at pH = 5.1 Gi (NH3) = 0.95, whereas at pH = 9.15 it increases to Gi = 3.1. Hydrogen sulphide is formed in airfree solutions, Gi (H2S) = 0.29, whereas in the presence of N2O it is reduced to Gi (H2S) = 0.10. Some probable reaction mechanisms are presented.     

Spectral sensitivity of cones in the goldfish, Carassius auratus

The spectral sensitivities of retinal cones isolated from goldfish (Carassius auratus) retinas were measured in the range 277-737 nm by recording membrane photocurrents with suction pipette electrodes (SPE). Cones were identified with lambda max (+/- S.D.) at 623 +/- 6.9 nm, 537 +/- 4.7 nm, 447 +/- 7.7 nm, and about 356 nm (three cells). Two cells (lambda max 572 and 576 nm) possibly represent genetic polymorphism. A single A2 template fits the alpha-band of P447(2), P537(2), and P623(2). HPLC analysis showed 4% retinal:96% 3-dehydroretinal. Sensitivity at 280 nm is nearly half that at the lambda max in the visible. The lambda max of the beta-band (in nm) is a linear function of the lambda max of the alpha-band and follows the same relation as found for A1-based cone pigments of a cyprinid fish.     

Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes

Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA adducts, genetic predisposition to cancer, and their interrelationships are under study in numerous laboratories. This report describes a modified 32P-postlabeling assay for the detection of polycyclic aromatic hydrocarbon-DNA adducts that uses immunoaffinity chromatography to enhance chemical specificity and quantitative reliability. The assay incorporates internal standards to determine direct molar ratios of adducts to unmodified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. High performance liquid chromatography is used to assure adequacy of DNA enzymatic digestion. The assay was validated using radiolabeled benzo(a)pyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby assessing all variables from enzymatic digestion to detection. Thirty-eight human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide-DNA adducts by immunoaffinity chromatography, high performance liquid chromatography, and synchronous fluorescence spectroscopy. A high correlation between the two assays was found (P = 0.006). The lung samples were then analyzed by the polymerase chain reaction for the presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione S-transferase mu (GST mu) genes. A positive association was identified for adduct levels and GST mu null genotypes (P = 0.038). No correlation was found between polycyclic aromatic hydrocarbon-adduct levels and CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related to adduct levels. Multivariate analysis indicated that only the GST mu genotype was associated with polycyclic aromatic hydrocarbon-DNA adduct levels. This work demonstrates that the 32P-postlabeling assay can be modified for chemically specific adduct detection and that it can be used in the assessment of potentially important genetic factors for cancer risk. The absence of a functional GST mu gene in humans is likely one such factor.     

Induction of apoptosis by the N-acetyl-galactosamine-specific toxic lectin from Viscum album L. is associated with a decrease of nuclear p53 and Bcl-2 proteins and induction of telomeric associations

Fluorescent probes serve as sensitive tools for obtaining structural and functional information in cellular systems. In spite of the high sensitivity provided by fluorescent reagents, cell surface receptors expressed in low numbers often escape detection with commonly used fluorescent probes. R-Phycoerythrin (R-PE), a molecule with a very high quantum yield, is often the reagent of choice for the detection of such low abundance events. We have developed streptavidin conjugates of two highly fluorescent 35-40 nm diameter polystyrene nanospheres, the green fluorescent FluoSpheres (Ex/Em 505/515) and red fluorescent TransFluoSpheres (Ex/Em 488/645). Like R-PE, the new reagents have peak excitations near 488 nm but differ in their emission maxima; 515 nm for the green nanospheres, 645 nm for the red nanospheres and 575 nm for R-PE. Hence the nanospheres are detected by flow cytometry in channels capable of detecting green (FL1) and red (FL3) fluorescence, while R-PE is detected in channel FL2. These nanospheres were tested for the detection of the sparsely expressed epidermal growth factor receptor (EGFR) of NIH-3T3 cells and the densely expressed EGFR of A431 cells. Results indicate that the nanosphere reagents are more sensitive than fluorescein-streptavidin and at least comparable in sensitivity to R-PE-streptavidin. The simultaneous use of these nanospheres with R-PE was also studied by concurrent staining of the CD3 and CD4 receptors in JURKAT cells. Labeling of CD4 receptors with streptavidin nanospheres and CD3 receptors with the R-PE-anti-CD3 conjugate confirmed the suitability of using the new nanospheres in combination with R-PE in multicolor flow cytometry experiments. This paper thus describes the use of alternative tools with detection sensitivity comparable to that of R-PE, but detected in different channels than R-PE, permitting their simultaneous use with R-PE.     

N2,7-bis(1-hydroxy-2-oxopropyl)-2'-deoxyguanosine: identical noncyclic adducts with 1,3-dichloropropene epoxides and methylglyoxal

cis- and trans-1,3-dichloropropene epoxides (1,3-D-epoxides) are proposed to be the penultimate or ultimate genotoxic metabolites of the major soil fumigant nematicide 1,3-dichloropropene. The 1, 3-D-epoxide isomers and the potential aldehydes from their degradation readily form adducts with 2'-deoxyguanosine (dGuo) but not with 2'-deoxyadenosine or 2'-deoxycytidine. The reaction of dGuo with the 1,3-D-epoxides (1:20 molar equiv) in phosphate buffer at pH 7.4 for 24 h at 37 degreesC results in complete conversion to four adducts that can be separated by HPLC with the same UV spectra and electrospray (ES)/MS molecular ion and fragmentation patterns. These adducts contain no chlorine and are identical to those obtained more rapidly with methylglyoxal in place of the 1,3-D-epoxides. The four isomeric methylglyoxal adducts with dGuo were proposed originally by others to be the cyclic adducts 1,N2-(1, 2-dihydroxy-2-methyl)ethano-dGuo, but they are reassigned here as the four diastereomers of the noncyclic bis adducts N2, 7-bis(1-hydroxy-2-oxopropyl)-dGuo. The assignments are based on HPLC/UV and HPLC/ES/MS experiments and 1H NMR spectral analysis of the first of the four adducts eluted with HPLC. Acid-catalyzed depurination converts the four dGuo derivatives to the two corresponding isomers of N2,7-bis(1-hydroxy-2-oxopropyl)guanine, assigned by ES/MS and 1H and 13C NMR. Although identical adducts are formed from dGuo with the 1,3-D-epoxides or methylglyoxal, the latter alpha,beta-dicarbonyl compound is not an intermediate in the reaction; instead, the 1,3-D-epoxides hydrolyze to 3-chloro-2-hydroxypropanal which adds to dGuo at N2 and N7. The adducts dehydrochlorinate, in a rate-limiting reaction, thereby giving the same end products obtained on direct reaction with methylglyoxal. Thus, 3-chloro-2-hydroxypropanal (not the 1, 3-D-epoxides or methylglyoxal) is the derivatizing agent for dGuo and therefore probably the mutagenic agent on 1,3-D bioactivation. On the basis of the dGuo model studied here, the DNA adducts of 1, 3-D and its epoxides may be the same as those with methylglyoxal [Vaca, C. E., Fang, J.-L., Conradi, M., and Hou, S.-M. (1994) Carcinogenesis 15, 1887-1894].     

Wittig reaction of unprotected D-aldopentoses with stabilized ylides: metal-ion effects

Each of the four D-aldopentoses (1-4) reacts in the unprotected form with Ph3PCHCO2Me (5) in THF to give stereoselectively the corresponding trans-alpha,beta-unsaturated C7 Wittig adducts, isolated as the corresponding 4,5,6,7-tetraacetates (7-10) or 4,5:6,7-di-O-isopropylidene derivatives (11-14). Concurrently formed are also bicyclic, 1,4-lactone derivatives, isolated as their 5,7-diacetates (15-18) or 5,7-O-isopropylidene derivatives (19), arising through intramolecular Michael addition from the initial acyclic adducts. Formation of the cyclized products is suppressed by incorporation of Cu(OAc)2 in the reaction mixture, permitting preparative isolation of the hept-3-enonate derivatives 7-10, useful as dienophiles in Diels-Alder carbocyclizations with chirality transfer. Optimized yields were 25% (D-ribo), 50% (D-arabino), 49% (D-xylo), and 61% (D-lyxo). Under Cu(OAc)2-catalyzed conditions, the formation of small proportions of 3,7-anhydroheptonic acid esters (21 and 23) was observed in the D-arabino and D-xylo series, but not with the other two pentoses.     

Acetylator phenotype, aminobiphenyl-hemoglobin adduct levels, and bladder cancer risk in white, black, and Asian men in Los Angeles, California

BACKGROUND: There is a large body of epidemiologic and experimental data that have identified a number of arylamines as human bladder carcinogens. Metabolic activation is required to biotransform these arylamines into their carcinogenic forms, and N-hydroxylation, which is catalyzed by the hepatic cytochrome P4501A2 isoenzyme, is generally viewed as the first critical step. On the other hand, the N-acetylation reaction, catalyzed by the hepatic N-acetyltransferase enzyme, represents a detoxification pathway for such compounds. The N-acetyltransferase enzyme is coded by a single gene displaying two phenotypes, slow and rapid acetylators. In the United States, cigarette smoking is a major cause of bladder cancer in men, and carcinogenic arylamines present in cigarette smoke are believed to be responsible for inducing bladder cancer in smokers. PURPOSE: Our purpose was to test the differences in three ethnic/racial groups for the prevalence of acetylator phenotypes and to ascertain whether slow acetylators actually have higher levels of activated arylamines in comparison with rapid acetylators. METHODS: One hundred thirty-three male residents of Los Angeles County who were either white, black, or Asian (Chinese or Japanese) and over the age of 35 years were assessed for their acetylator phenotype and levels of 3- and 4-aminobiphenyl (ABP) hemoglobin adducts. Subjects were either lifetime nonsmokers (n = 72) or current cigarette smokers of varying intensity (n = 61). RESULTS: The proportion of slow acetylators was highest among whites (54%), intermediate among blacks (34%), and lowest among Asians (14%). Similarly, geometric mean levels of both 3- and 4-ABP-hemoglobin adducts were highest in whites (1.80 and 49.2 pg/g hemoglobin [Hb], respectively), intermediate in blacks (1.54 and 38.5 pg/g Hb), and lowest in Asians (0.73 and 36.0 pg/g Hb). As expected, cigarette smokers had significantly higher mean levels of both 3- and 4-ABP-hemoglobin adducts relative to nonsmokers, and the levels increased with the number of cigarettes smoked per day (P < .0005 for both adducts). Slow acetylators consistently exhibited higher mean levels of ABP-hemoglobin adducts relative to rapid acetylators, independent of race and level of smoking. CONCLUSION: The present cross-sectional survey supports acetylation phenotype as an important determinant of bladder cancer risk and a possible major factor in the varying bladder cancer risk among whites, blacks, and Asians.     

Chemiluminescence detection in integrated post-separation reactors for microchip-based capillary electrophoresis and affinity electrophoresis

Chemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post-separation detection scheme for microchip-based capillary electrophoresis. An integrated injector, separator and post-separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response. In devices etched 10 microm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP-F1. In devices etched 40 microm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')2 fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG was performed on-chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 microg/mL) with increasing mouse IgG (0-60 microg/mL). When microperoxidase was used as an internal standard, the R2 value of a linear least-squares fit was 0.9867, and the relative errors in the slope and intercept were +/- 5.8 and +/- 1.3 %, respectively.     

Interferon-gamma and retinoic acid down-regulate N-myc in neuroblastoma through complementary mechanisms of action

A method is described for the assay of the major malondialdehyde-deoxyguanosine adduct (M1G) based on immunoaffinity purification and gas chromatography/electron capture/negative chemical ionization/mass spectrometry. A stable isotope of M1G-deoxyribose ([2H2]M1G-dR) was used as an internal standard. Recovery of internal standard throughout the entire assay procedure was approximately 40%. The assay showed a linear response over a range of 10-1000 pg of M1G-dR and was verified by analysis of a synthetic. M1G-containing oligomer. The limit of detection in biological samples was 100 fmol/sample, corresponding to 3 adducts/10(8) bases for 1 mg of DNA. DNA was isolated from the blood of 10 healthy human donors, and M1G levels were measured. A mean value of 6.2 +/- 1.2 adducts/10(8) bases was obtained, with no obvious differences bases on age or cigarette smoking. A small, but statistically significant difference was observed between the levels in females (5.1 +/- 0.4 adducts/10(8) bases) and males 6.7 +/- 1.1 adducts/10(8) bases). The presence of M1G in leukocyte DNA was further verified by analysis using liquid chromatography/electrospray ionization mass spectrometry.