Biomedical subjects

D R Doerge

Publications and source records attributed to D R Doerge.

At least 37 records · Page 2Linked to original sources

PubMed2000

Quantification of etheno-DNA adducts using liquid chromatography, on-line sample processing, and electrospray tandem mass spectrometry.

Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 10(8) normal nucleotides from 100 microg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethane-treated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.

Animals↗

PubMed2000

Mass spectrometric determination of Genistein tissue distribution in diet-exposed Sprague-Dawley rats.

Genistein, the principal soy isoflavone, was administered in the diet to male and female Sprague-Dawley rats as part of a multigeneration study of potential endocrine modulation. The rats were exposed to genistein in utero, through maternal milk, and as adults through postnatal d 140 via essentially isoflavone-free feed (approximately 0.5 microg/g) fortified at 5, 100 and 500 microg/g with genistein aglycone. Analytical methods based on liquid chromatography, mass spectrometry and the use of deuterated genistein were developed and validated for use in measuring genistein in serum and tissues. Pharmacokinetic analysis of serum genistein showed a significant difference (P < 0.001) in the elimination half-life and area under the concentration-time curve between male [2.97 +/- 0.14 h and 22.3 +/- 1.2 micromol/(L. h), respectively] and female rats [4.26 +/- 0.29 h and 45.6 +/- 3.1 micromol/(L. h), respectively, +/- SEM]. Endocrine-responsive tissues including brain, liver, mammary, ovary, prostate, testis, thyroid and uterus showed significant dose-dependent increases in total genistein concentration. Female liver contained the highest amount of genistein (7.3 pmol/mg tissue) and male whole brain contained the least (0.04 pmol/mg). The physiologically active aglycone form was present in tissues at fractions up to 100%, and the concentration was always greater than that observed in serum in which conjugated forms predominated (95-99%). These results for measured amounts of genistein, present as aglycone and conjugates, in putative target tissues provide a link with other studies in which blood concentrations and physiologic effects of genistein are measured.

Animals↗

PubMed2000

Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry.

Soybean products containing isoflavones are widely consumed in Western and Asian diets for putative health benefits, but adverse effects are also possible. The conjugated forms of isoflavones present in a soy nutritional supplement (predominately acetyl glucosides) and in blood from two human volunteers after consuming the supplement (7- and 4'-glucuronides and sulfates) were identified using liquid chromatography coupled with electrospray/tandem mass spectrometry. Circulating conjugates of genistein and daidzein were quantified using selective enzymatic hydrolysis and deuterated internal standards for liquid chromatography-electrospray/mass spectrometry. The levels of isoflavone glucuronides were much greater than the corresponding sulfates or aglycones. The substrate activities of genistein and daidzein were evaluated with recombinant human UDP glucuronosyl transferase (UGT) and sulfotransferase (SULT) by using enzyme kinetics. The SULTs 1A1*2, 1E, and 2A1 catalyzed formation of a single genistein sulfate; however, SULTs 1A2*1 and 1A3 had no observed activity. None of the SULTs showed activity with daidzein. Although several UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) catalyzed 7- and 4'-glucuronidation of genistein or daidzein, the UGT 1A10 isoform, which is found in human colon but not liver, was found to be specific for genistein. Glucuronidation of only genistein was observed in human colon microsomes, although nearly equal activity was observed for daidzein in human liver and kidney microsomes. These findings suggest a prominent role for glucuronidation of genistein in the intestine concomitant with absorption, although hepatic glucuronidation of absorbed genistein and daidzein aglycones is also likely.

Animals↗

PubMed2000

A simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols.

A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M(-1)) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.

Adult↗

PubMed1999

Inactivation of Coprinus cinereus peroxidase by 4-chloroaniline during turnover: comparison with horseradish peroxidase and bovine lactoperoxidase.

The peroxidase from Coprinus cinereus (CPX) catalyzed oxidative oligomerization of 4-chloroaniline (4-CA) forming several products: N-(4-chlorophenyl)-benzoquinone monoamine (dimer D), 4,4'-dichloroazobenzene (dimer E); 2-(4-chloroanilino)-N-(4-chlorophenyl)-benzoquinone (trimer F); 2-amino-5-chlorobenzoquinone-di-4-chloroanil (trimer G); 2-(4-chloroanilino)-5-hydroxybenzoquinone-di-4-chloroanil (tetramer H) and 2-amino-5-(-4-chlroanilino)-benzoquinone-di-4-chloroanil (tetramer 1). In the presence of 4-CA and H2O2, CPX was irreversibly inactivated within 10 min. Inactivation of CPX in the presence of H2O2 was a time-dependent, first-order process when the concentration of 4-CA was varied between 0 and 2.5 mM. The apparent dissociation constant (Ki) for CPX and 4-CA was 0.71 mM. The pseudo-first order rate constant for inactivation (k(inact)), was 1.15 x 10(-2) s(-1). Covalent incorporation of 20 mole 14C-4-CA per mole of inactivated CPX was observed. The partition ratio was about 2200 when either 4-CA or H2O2 was used as the limiting substrate. These results show that 4-CA is a metabolically activated inactivator (i.e. a suicide substrate). Unmodified heme and hydroxymethyl heme were isolated from native, 4-CA-inactivated and H2O2-incubated CPX. Inactivation resulted in significant losses in both heme contents. Analysis of tryptic peptides from 4-CA-inactivated CPX by MALDI-TOF/ MS and UV-VIS spectrophotometry suggested that trimer G and tetramer H were the major 4-CA derivatives that were covalently bound, including to a peptide (MGDAGF-SPDEVVDLLAAHSLASQEGLNSAIFR) containing the heme binding site. These studies show that heme destruction and covalent modification of the polypeptide chain are both important for the inactivation of CPX. These results were compared with similar studies on 4-CA-inactivated horseradish peroxidase (HRP) and bovine lactoperoxidase (LPO) during the oxidation of 4-CA.

Amino Acid Sequence↗

PubMed1999

Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice.

Malachite green, an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in commercial fish hatcheries. Malachite green is reduced to and persists as leucomalachite green in the tissues of fish. Female and male B6C3F1 mice and Fischer 344 rats were fed up to 1200 ppm malachite green or 1160 ppm leucomalachite green for 28 days to determine the toxicity and metabolism of the dyes. Apoptosis in the transitional epithelium of the urinary bladder occurred in all mice fed the highest dose of leucomalachite green. This was not observed with malachite green. Hepatocyte vacuolization was present in rats administered malachite green or leucomalachite green. Rats given leucomalachite green also had apoptotic thyroid follicular epithelial cells. Decreased T4 and increased TSH levels were observed in male rats given leucomalachite green. A comparison of adverse effects suggests that exposure of rats or mice to leucomalachite green causes a greater number of and more severe changes than exposure to malachite green. N-Demethylated and N-oxidized malachite green and leucomalachite green metabolites, including primary arylamines, were detected by high performance liquid chromatography/mass spectrometry in the livers of treated rats. 32P-Postlabeling analyses indicated a single adduct or co-eluting adducts in the liver DNA. These data suggest that malachite green and leucomalachite green are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to malachite green.

Aniline Compounds↗

PubMed1999

An FDA review of sulfamethazine toxicity.

Recently, changes have been proposed in the criteria historically used in the evaluation of the applicability to humans of some of the results obtained from the rodent carcinogenicity bioassay data. These questions center on the suitability of the rodent model for agents that exert their toxic effects via specific enzyme interactions and endocrine mechanisms which appear to be inoperative within humans. Within the U.S. Food and Drug Administration (FDA), this issue has been brought to the forefront of concern with the recent application for a New Animal Drug Application for sulfamethazine (SMZ). A panel of FDA experts from the National Center for Toxicological Research (NCTR), the Center for Veterinary Medicine (CVM), and the Center for Food Safety and Applied Nutrition has reviewed the sum of the scientific evidence available on the toxicology of SMZ. They noted that, in previous feeding studies at NCTR, high doses of SMZ were associated with significant incidences of thyroid tumors in mice and rats. The panel also notes that the tumorigenic activity of SMZ in rodents was due to its goitrogenic activity, resulting in constant stimulation of the thyroid by TSH. Humans, on the other hand, were found to be insensitive to the SMZ-like inhibition of thyroid function. Further, apart from X-irradiation and radioactive iodine, there are no other physical or chemical agents known to cause thyroid tumors in humans. Thus, the expert panel concludes that the best scientific information available indicates that elevated levels of TSH and the consequent thyroid tumors would not be produced under approved use conditions of SMZ. This conclusion is in agreement with recommendations made by three other panels, viz. the World Health Organization, the U.S. Environmental Protection Agency, and CVM, which also evaluated the public health risk of SMZ.

Animals↗

PubMed1999

Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS.

Genistein is the principal soy isoflavone to which the putative beneficial effects of soy consumption have been attributed; however, the possibility of adverse biological effects (e.g., estrogenic, antithyroid) has also been raised. This paper describes development and validation of a simple and sensitive analytical method for the determination of genistein in the blood of rats receiving dietary genistein (<0.5-1250 microg of genistein aglycone/g of chow). The method uses serum/plasma deproteination, liquid-liquid extraction, deuterated genistein and daidzein internal standards, isocratic LC separation, and electrospray mass spectrometric quantification using selected ion monitoring. Extraction efficiency is approximately 85%, the detection limits for genistein and daidzein from 50 microL of rat blood are approximately 5 nM, and the limit of quantification is approximately 15 nM. Interassay precision (relative standard deviation 4.5-4.6%) and intraassay precision (3.3-6.7%) were determined from replicate analysis of a spiked control and an incurred serum sample. The distribution of conjugated and unconjugated forms of genistein in the blood of rats was determined using selective enzyme hydrolysis. The glucuronide was the predominant metabolite (>90%), and only small amounts of the sulfate conjugate and the aglycone were observed at all dose levels. No evidence for additional metabolites was obtained. The 7- and 4'-glucuronide conjugates of genistein were identified using electrospray mass spectrometry and (1)H NMR. Total blood genistein ranged from <15 nM in animals fed soy-free control diet to as high as 8.9 microM in male rats fed 1250 microg of genistein/g of chow and encompasses blood isoflavone levels observed in humans consuming a typical Asian diet and nutritional supplements (0.1-1 microM) and infants consuming soy formulas (2-7 microM).

Animals↗

PubMed1999

Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard.

32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P-postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N-Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 +/- 0.8 adducts/10(8) nucleotides (mean +/- SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 +/- 1.7 dG-C8-4-ABP/10(8) nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG-C8-4-ABP/10(8) nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 +/- 26 and 63 +/- 20 dG-C8-4-ABP/10(8) nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.

Aminobiphenyl Compounds↗

PubMed1999

Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC-ES-MS.

Electrospray mass spectrometry (ES-MS) is a powerful tool for analysis of carcinogen-adducted DNA. In this study, we developed a quantitative isotope dilution method for analysis of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4-ABP), the principal nucleoside adduct derived from enzymatic hydrolysis of 4-aminobiphenyl (4-ABP)-modified DNA. The method used column switching valves to perform on-line sample concentration and cleanup, which permitted direct analysis of enzymatic DNA hydrolysates using narrow-bore liquid chromatography (LC). ES-MS detection was performed using a single quadrupole instrument by monitoring M+H+ and two fragment ions characteristic for dG-C8-4-ABP, along with M+H+ and a fragment ion for the deuterated internal standard. The detection limit for dG-C8-4-ABP in DNA hydrolysates was approximately 10 pg on-column, equivalent to 0.7 dG-C8-4-ABP adducts in 10(7) normal nucleotides for a sample containing 100 microg DNA. The method was applied to the analysis of calf thymus DNA modified in vitro through reaction with N-hydroxy-4-ABP and of hepatic DNA isolated from mice treated in vivo with two dose levels of 4-ABP.

Aminobiphenyl Compounds↗

PubMed1998

Fungal metabolism of nitrofluoranthenes.

Metabolism of 2-nitrofluoranthene (2-NFA), one of the most abundant and genotoxic environmental pollutants in air, and of a mixture of 2-nitrofluoranthene and 3-nitrofluoranthene (3-NFA) was studied using (1) the fungus Cunninghamella elegans ATCC 36112 and (2) rat liver microsomes. The fungal metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and identified by 1H nuclear magnetic resonance (NMR) spectrometry, ultraviolet (UV)-visible spectroscopy, and online atmospheric-pressure chemical ionization/mass spectrometry (APCI/MS). The fungus metabolized 82% of 2-nitro-[3H]-fluoranthene to 2-nitrofluoranthene 8-sulfate and 2-nitrofluoranthene 9-sulfate. Metabolism of a mixture of 2- and 3-nitrofluoranthene by C. elegans similarly produced 2-nitrofluoranthene 8- and 9-sulfate and 3-nitrofluoranthene 8- and 9-sulfate as major metabolites. In addition, a glucoside conjugate of 3-hydroxy-2-nitrofluoranthene was tentatively identified by APCI/MS analysis. When rat liver microsomes were incubated with a mixture of 2- and 3-nitrofluoranthene for 1 h, in addition to the trans-7,8- and 9,10-dihydrodiols reported previously for 2-nitrofluoranthene, several novel metabolites were produced including 2-nitrofluoranthene trans-4,5-dihydrodiol and 2-nitrofluoranthene trans-8,9-dihydrodiol, the trans-4,5-dihydrodiol of 3-nitrofluoranthene, and phenolic products of both 2- and 3-nitrofluoranthene. The fungal metabolism of the 2- and 3-nitrofluoranthene mixture was similar to the metabolism of individual nitrofluoranthenes; however, the mammalian metabolism of the nitrofluoranthene mixture showed differences in regioselectivity at positions C4, C5, C8, and C9.

Air Pollutants↗

PubMed1998

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry.

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.

Aniline Compounds↗

PubMed1998

Mass spectrometric analysis of 2-deoxyribonucleoside and 2'-deoxyribonucleotide adducts with aldehydes derived from lipid peroxidation.

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of alpha, beta-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3'- and 5'-monophosphate; 3',5'-bisphosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans.

Aldehydes↗

PubMed1998

Mechanism for inhibition of thyroid peroxidase by leucomalachite green.

The triphenylmethane dye, malachite green (MG), is used to treat and prevent fungal and parasitic infections in the aquaculture industry. It has been reported that the reduced metabolite of MG, leucomalachite green (LMG), accumulates in the tissues of fish treated with MG. MG is structurally related to other triphenylmethane dyes (e.g., gentian violet and pararosaniline) that are carcinogenic in the liver, thyroid, and other organs of experimental animals. The ability of LMG to inhibit thyroid peroxidase (TPO), the enzyme that catalyzes the iodination and coupling reactions required for thyroid hormone synthesis, was determined in this study. LMG inhibited TPO-catalyzed tyrosine iodination (half-maximal inhibition at ca. 10 microM). LMG also inhibited the TPO-catalyzed formation of thyroxine in low-iodine human goiter thyroglobulin (half-maximal inhibition at ca. 10 microM) using a model system that measures simultaneous iodination and coupling. Direct inhibition of the coupling reaction by LMG was shown using a coupling-only system containing chemically preiodinated thyroglobulin as the substrate. Incubation of LMG with TPO, iodide, and tyrosine in the presence of a H2O2-generating system yielded oxidation products that were identified by using on-line LC/APCI-MS as desmethyl LMG, 2desmethyl LMG, 3desmethyl LMG, MG, and MG N-oxide. Similar products from LMG were observed in incubations with TPO and H2O2 alone. These findings suggest that the anti-thyroid effects (increased serum thyroid-stimulating hormone and decreased serum thyroxine) observed in rats treated with LMG result from blockade of hormone synthesis through alternate substrate inhibition and that chronic exposure could cause thyroid follicular cell tumors through a hormonal mechanism. The observed TPO-catalyzed oxidative demethylation of LMG to a primary arylamine also suggests a genotoxic mechanism for tumor formation is possible.

Aniline Compounds↗

PubMed1998

An improved 32P-postlabeling/high-performance liquid chromatography method for the analysis of the malondialdehye-derived 1, N2-propanodeoxyguanosine DNA adduct in animal and human tissues.

Malondialdehyde (MDA) is a major lipid peroxidation product that is mutagenic and tumorigenic. The MDA-modified DNA adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1, 2-alpha]purin-10(3H)-one (M1G), has been detected in human tissues and may be a marker of human cancer risk. In this paper, we describe an improved 32P-postlabeling/HPLC method for sensitive detection and quantitation of this MDA-modified 2'-deoxyribonucleotide adduct. Specific improvements include (i) unequivocal structural identification of the postlabeling products, both the 3', 5'-bisphosphate of M1G (MDA-3',5'-dGDP) and the 5'-monophosphate of M1G (MDA-5'-dGMP); (ii) efficient separation of the 32P-postlabeling products by HPLC; and (iii) the incorporation of a synthetically prepared MDA-modified DNA (or the 3'-monophosphate of M1G) with a known modification level as an internal standard. This improved quantitative methodology provides high intra- and inter-assay reproducibility and has been applied to the analysis of this adduct in rodent and human samples.

Adult↗

PubMed1997

Anti-thyroid isoflavones from soybean: isolation, characterization, and mechanisms of action.

The soybean has been implicated in diet-induced goiter by many studies. The extensive consumption of soy products in infant formulas and in vegetarian diets makes it essential to define the goitrogenic potential. In this report, it was observed that an acidic methanolic extract of soybeans contains compounds that inhibit thyroid peroxidase- (TPO) catalyzed reactions essential to thyroid hormone synthesis. Analysis of the soybean extract using HPLC, UV-VIS spectrophotometry, and LC-MS led to identification of the isoflavones genistein and daidzein as major components by direct comparison with authentic standard reference isoflavones. HPLC fractionation and enzymatic assay of the soybean extract showed that the components responsible for inhibition of TPO-catalyzed reactions coeluted with daidzein and genistein. In the presence of iodide ion, genistein and daidzein blocked TPO-catalyzed tyrosine iodination by acting as alternate substrates, yielding mono-, di-, and triiodoisoflavones. Genistein also inhibited thyroxine synthesis using iodinated casein or human goiter thyroglobulin as substrates for the coupling reaction. Incubation of either isoflavone with TPO in the presence of H2O2 caused irreversible inactivation of the enzyme; however, the presence of iodide ion in the incubations completely abolished the inactivation. The IC50 values for inhibition of TPO-catalyzed reactions by genistein and daidzein were ca. 1-10 microM, concentrations that approach the total isoflavone levels (ca. 1 microM) previously measured in plasma from humans consuming soy products. Because inhibition of thyroid hormone synthesis can induce goiter and thyroid neoplasia in rodents, delineation of anti-thyroid mechanisms for soy isoflavones may be important for extrapolating goitrogenic hazards identified in chronic rodent bioassays to humans consuming soy products.

Antithyroid Agents↗

PubMed1997

Identification of the colored guaiacol oxidation product produced by peroxidases.

Oxidation of guaiacol by peroxidases in the presence of H2O2 is the basis for a widely used colorimetric assay. However, the nature of the assay product, which has an absorption maximum around 470 nm, had not been determined. In the present study, we combined HPLC with a rapid scanning uv-visible detector and observed a single product with a spectrum identical to the assay product from the reaction catalyzed by lactoperoxidase. Analysis of the reaction product using on-line HPLC with atmospheric pressure chemical ionization detection (LC-APCI/MS) yielded a mass spectrum consistent with 3,3 '-dimethoxy-4,4'-biphenylquinone. A minor reaction product was observed with mass spectrum consistent with 3,3'-dimethoxy-4,4'-dihydroxybiphenyl. The presence of a catechol impurity in guaiacol was previously shown to yield an additional product from peroxidase-mediated oxidation based on its visible absorption (Taurog et al., 1992 Anal. Biochem. 205, 271-277). When such an incubation mixture was analyzed using LC-APCI/MS, a product with mass spectrum consistent with 3-methoxy-2',3',4-trihydroxybiphenyl was observed. Identification of such a heterodimeric product supports the previously proposed mechanism for catechol interference in the guaiacol assay as well as the radical nature of peroxidase-catalyzed oxidation of phenols.

Biphenyl Compounds↗

PubMed1997

Identification of ceramides in human cells using liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry.

Ceramides are intermediates in the biosynthesis of membrane sphingolipids. These biomolecules are also important as second messengers in signal transduction pathways controlling cell growth. We have developed two reversed-phase high pressure liquid chromatography (RPHPLC) techniques for identification and quantification of ceramides from mammalian cells. One method was based on atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) detection of ceramides and had the advantage of requiring minimal sample preparation, yielding significant structural information, and affording high sensitivity. The second method relied on perbenzoylation of the ceramides and detection at 230 nm. The predominant ceramides detected in the human leukemic HL-60 cell were N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine. When selected ion monitoring was used with RPHPLC/APCI-MS, approximately 2.2 pmol N-(palmitoyl)-sphingosine and 1.7 pmol N-(nervonyl)-sphingosine were observed in an extract from 40,000 HL-60 cells. Perbenzoylation with benzoyl chloride permitted RPHPLC separation and 230 nm UV absorbance detection of the trisbenzoyl derivatives of sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine in the HL-60 cells. These results demonstrate the utility of utilizing two different methods coupled with APCI-MS for the quantification and identification of ceramides in biological samples.

Benzyl Compounds↗