Biomedical subjects

F F Hsu

Publications and source records attributed to F F Hsu.

At least 55 records · Page 3Linked to original sources

PubMed1998

Hemoglobin Raleigh as the cause of a falsely increased hemoglobin A1C in an automated ion-exchange HPLC method.

Irreversible glycation of the hemoglobin A0 (HbA0) beta chain leads to the production of HbA1C, which can be used to monitor long-term blood glucose control in patients with diabetes mellitus. HbA1C is less positively charged than nonglycated HbA0, and this decrease in charge is the basis of ion-exchange and electrophoretic methods that measure HbA1C. We recently identified a sample that appeared to contain 46% HbA1C by an automated ion-exchange HPLC method (Bio-Rad Variant) but only 3.8% by an immunoinhibition latex agglutination method. A combination of traditional and mass spectrometric protein analysis and genomic DNA analysis of the Hb beta chain and genes revealed that the patient was heterozygotic for Hb-Raleigh, a variant containing a valine-->alanine substitution at position 1 of the beta chain. The amino-terminal alanine in this variant Hb is posttranslationally modified by acetylation, leading to a charge difference similar to glycation and making the behavior of HbA1C and Hb Raleigh virtually identical in the ion-exchange HPLC method. This observation suggests that it is important to confirm HbA1C values in excess of 15%, especially if they are not consistent with the clinical picture, by an independent HbA1C method such as immunoassay or boronic acid affinity chromatography. However, for this particular variant Hb, even these latter methods might be misleading, because the acetylated N-terminal amino acid of the Hb-Raleigh beta chain cannot be glycated.

Aged↗

PubMed1997

Benzidine-DNA adduct levels in human peripheral white blood cells significantly correlate with levels in exfoliated urothelial cells.

In a cross-sectional study of 33 workers exposed to benzidine and benzidine dyes and 15 non-exposed controls, we previously reported that exposure status and internal dose of benzidine metabolites were strongly correlated with the levels of specific benzidine-DNA adducts in exfoliated urothelial cells. We also evaluated DNA adduct levels in peripheral white blood cells (WBC) of a subset of 18 exposed workers and 7 controls selected to represent a wide range of adducts in exfoliated urothelial cells. Samples were coded and then DNA was analyzed using 32P-postlabeling, along with n-butanol extraction. One adduct, which co-chromatographed with a synthetic N-(3'-phospho-deoxyguanosin-8-yl)-N'-acetylbenzidine standard, predominated in those samples with adducts present. The median level (range) of this adduct in WBC DNA was 194.4 (3.2-975) RAL x 10(9) in exposed workers and 1.4 (0.1-6.4) in the control subjects (p = 0.0002, Wilcoxon Rank Sum Test). There was a striking correlation between WBC and exfoliated urothelial cell adduct levels (Pearson r = 0.84, p < 0.001) among exposed subjects. In addition, the sum of urinary benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine correlated with the levels of this adduct in both tissues. This is the first study in humans to show a relationship for a specific carcinogen adduct in a surrogate tissue and in urothelial cells, the target for urinary bladder cancer.

Adult↗

PubMed1997

p-Hydroxyphenylacetaldehyde, the major product of L-tyrosine oxidation by the myeloperoxidase-H2O2-chloride system of phagocytes, covalently modifies epsilon-amino groups of protein lysine residues.

Activated human phagocytes employ the myeloperoxidase-H2O2-Cl- system to convert L-tyrosine to p-hydroxyphenylacetaldehyde (pHA). We have explored the possibility that pHA covalently reacts with proteins to form Schiff base adducts, which may play a role in modifying targets at sites of inflammation. Because Schiff bases are labile to acid hydrolysis, prior to analysis the adducts were rendered stable by reduction with NaCNBH3. Purified pHA reacted with Nalpha-acetyllysine, an analog of protein lysine residues. The reduced reaction product was identified as Nalpha-acetyl-Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine by 1H NMR spectroscopy and mass spectrometry. The compound Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine (pHA-lysine) was likewise identified in acid hydrolysates of bovine serum albumin (BSA) that were first exposed to myeloperoxidase, H2O2, L-tyrosine, and Cl- and then reduced with NaCNBH3. Other halides (F-, Br-, I-) and the pseudohalide SCN- could not replace Cl- as a substrate in the myeloperoxidase-H2O2-L-tyrosine system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, L-tyrosine, and reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was incubated with L-tyrosine and HOBr, peroxynitrite, hydroxyl radical, or a variety of other peroxidases, indicating that the aldehyde-protein adduct was selectively produced by HOCl. Human neutrophils activated in the presence of tyrosine also modified BSA lysine residues. pHA-lysine formation required L-tyrosine and cell activation; it was inhibited by peroxidase inhibitors and catalase, implicating myeloperoxidase and H2O2 in the reaction pathway. pHA-lysine was detected in inflamed human tissues that were reduced, hydrolyzed, and then analyzed by mass spectrometry, indicating that the reaction of pHA with proteins may be of physiological importance. These observations raise the possibility that the identification of pHA-lysine in tissues will pinpoint targets where phagocytes inflict oxidative damage in vivo.

Acetaldehyde↗

PubMed1997

Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques.

Lipoprotein oxidation has been implicated in the pathogenesis of atherosclerosis. However, the physiologically relevant pathways mediating oxidative damage have not yet been identified. Three potential mechanisms are tyrosyl radical, hydroxyl radical, and redox active metal ions. Tyrosyl radical forms o,o'-dityrosine cross-links in proteins. The highly reactive hydroxyl radical oxidizes phenylalanine residues to o-tyrosine and m-tyrosine. Metal ions oxidize low density lipoprotein (LDL) by poorly understood pathways. To explore the involvement of tyrosyl radical, hydroxyl radical, and metal ions in atherosclerosis, we developed a highly sensitive and quantitative method for measuring levels of o, o'-dityrosine, o-tyrosine, and m-tyrosine in proteins, lipoproteins, and tissue, using stable isotope dilution gas chromatography-mass spectrometry. We showed that o,o'-dityrosine was selectively produced in LDL oxidized with tyrosyl radical. Both o-tyrosine and o, o'-dityrosine were major products when LDL was oxidized with hydroxyl radical. Only o-tyrosine was formed in LDL oxidized with copper. Similar profiles of oxidation products were observed in bovine serum albumin oxidized with the three different systems. Applying these findings to LDL isolated from human atherosclerotic lesions, we detected a 100-fold increase in o,o'-dityrosine levels compared to those in circulating LDL. In striking contrast, levels of o-tyrosine and m-tyrosine were not elevated in LDL isolated from atherosclerotic tissue. Analysis of fatty streaks revealed a similar pattern of oxidation products; compared with normal aortic tissue, there was a selective increase in o,o'-dityrosine with no change in o-tyrosine. The detection of a selective increase of o,o'-dityrosine in LDL isolated from vascular lesions is consistent with the hypothesis that oxidative damage in human atherosclerosis is mediated in part by tyrosyl radical. In contrast, these observations do not support a role for free metal ions as catalysts of LDL oxidation in the artery wall.

Animals↗

PubMed1997

Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to convert hydroxy-amino acids into glycolaldehyde, 2-hydroxypropanal, and acrolein. A mechanism for the generation of highly reactive alpha-hydroxy and alpha,beta-unsaturated aldehydes by phagocytes at sites of inflammation.

Reactive aldehydes derived from reducing sugars and lipid peroxidation play a critical role in the formation of advanced glycation end (AGE) products and oxidative tissue damage. We have recently proposed another mechanism for aldehyde generation at sites of inflammation that involves myeloperoxidase, a heme enzyme secreted by activated phagocytes. We now demonstrate that human neutrophils employ the myeloperoxidase-H202-chloride system to produce alpha-hydroxy and alpha,beta-unsaturated aldehydes from hydroxy-amino acids in high yield. Identities of the aldehydes were established using mass spectrometry and high performance liquid chromatography. Activated neutrophils converted L-serine to glycolaldehyde, an alpha-hydroxyaldehyde which mediates protein cross-linking and formation of Nepsilon-(carboxymethyl)lysine, an AGE product. L-Threonine was similarly oxidized to 2-hydroxypropanal and its dehydration product, acrolein, an extremely reactive alpha,beta-unsaturated aldehyde which alkylates proteins and nucleic acids. Aldehyde generation required neutrophil activation and a free hydroxy-amino acid; it was inhibited by catalase and heme poisons, implicating H202 and myeloperoxidase in the cellular reaction. Aldehyde production by purified myeloperoxidase required H202 and chloride, and was mimicked by reagent hypochlorous acid (HOCl) in the absence of enzyme, suggesting that the reaction pathway involves a chlorinated intermediate. Collectively, these results indicate that the myeloperoxidase-H202-chloride system of phagocytes converts free hydroxy-amino acids into highly reactive alpha-hydroxy and alpha,beta-unsaturated aldehydes. The generation of glycolaldehyde, 2-hydroxypropanal, and acrolein by activated phagocytes may thus play a role in AGE product formation and tissue damage at sites of inflammation.

Acetaldehyde↗

PubMed1997

Acidic urine pH is associated with elevated levels of free urinary benzidine and N-acetylbenzidine and urothelial cell DNA adducts in exposed workers.

We evaluated the influence of urine pH on the proportion of urinary benzidine (BZ) and N-acetylbenzidine present in the free, unconjugated state and on exfoliated urothelial cell DNA adduct levels in 32 workers exposed to BZ in India. Postworkshift urine pH was inversely correlated with the proportions of BZ (r = -0.78; P < 0.0001) and N-acetylbenzidine (r = -0.67; P < 0.0001) present as free compounds. Furthermore, the average of each subject's pre- and postworkshift urine pH was negatively associated with the predominant urothelial DNA adduct (P = 0.0037, adjusted for urinary BZ and metabolites), which has been shown to cochromatograph with a N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine adduct standard. Controlling for internal dose, individuals with urine pH < 6 had 10-fold higher DNA adduct levels compared to subjects with urine pH > or = 7. As reported previously, polymorphisms in NAT1, NAT2, and GSTM1 had no impact on DNA adduct levels. This is the first study to demonstrate that urine pH has a strong influence on the presence of free urinary aromatic amine compounds and on urothelial cell DNA adduct levels in exposed humans. Because there is evidence that acidic urine has a similar influence on aromatic amines derived from cigarette smoke, urine pH, which is influenced by diet, may be an important susceptibility factor for bladder cancer caused by tobacco in the general population.

Analysis of Variance↗

PubMed1996

Molecular chlorine generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes converts low density lipoprotein cholesterol into a family of chlorinated sterols.

Oxidation of low density lipoprotein (LDL) may be of critical importance in triggering the pathological events of atherosclerosis. Myeloperoxidase, a heme protein secreted by phagocytes, is a potent catalyst for LDL oxidation in vitro, and active enzyme is present in human atherosclerotic lesions. We have explored the possibility that reactive intermediates generated by myeloperoxidase target LDL cholesterol for oxidation. LDL exposed to the myeloperoxidase-H2O2-Cl- system at acidic pH yielded a family of chlorinated sterols. The products were identified by mass spectrometry as a novel dichlorinated sterol, cholesterol alpha-chlorohydrin (6beta-chlorocholestane-(3beta,5alpha)-diol), cholesterol beta-chlorohydrin (5alpha-chlorocholestane-(3beta, 6beta)-diol), and a structurally related cholesterol chlorohydrin. Oxidation of LDL cholesterol by myeloperoxidase required H2O2 and Cl-, suggesting that hypochlorous acid (HOCl) was an intermediate in the reaction. However, HOCl failed to generate chlorinated sterols under chloride-free conditions. Since HOCl is in equilibrium with molecular chlorine (Cl2) through a reaction which requires Cl- and H+, this raised the possibility that Cl2 was the actual chlorinating intermediate. Consonant with this hypothesis, HOCl oxidized LDL cholesterol in the presence of Cl- and at acidic pH. Moreover, in the absence of Cl- and at neutral pH, Cl2 generated the same family of chlorinated sterols as the myeloperoxidase-H2O2-Cl- system. Finally, direct addition of Cl2 to the double bond of cholesterol accounts for dichlorinated sterol formation by myeloperoxidase. Collectively, these results indicate that Cl2 derived from HOCl is the chlorinating intermediate in the oxidation of cholesterol by myeloperoxidase. Our observations suggest that Cl2 generation in acidic compartments may constitute one pathway for oxidation of LDL cholesterol in the artery wall.

Chloramines↗

PubMed1996

Human neutrophils employ chlorine gas as an oxidant during phagocytosis.

Reactive oxidants generated by phagocytes are of central importance in host defenses, tumor surveillance, and inflammation. One important pathway involves the generation of potent halogenating agents by the myeloperoxidase-hydrogen peroxide-chloride system. The chlorinating intermediate in these reactions is generally believed to be HOCl or its conjugate base, ClO-. However, HOCl is also in equilibrium with Cl2, raising the possibility that Cl2 executes oxidation/ halogenation reactions that have previously been attributed to HOCl/ClO-. In this study gas chromatography-mass spectrometric analysis of head space gas revealed that the complete myeloperoxidase-hydrogen peroxide-chloride system generated Cl2. In vitro studies demonstrated that chlorination of the aromatic ring of free L-tyrosine was mediated by Cl2 and not by HOCl/ClO-. Thus, 3-chlorotyrosine serves as a specific marker for Cl2-dependent oxidation of free L-tyrosine. Phagocytosis of L-tyrosine encapsulated in immunoglobulin- and complement-coated sheep red blood cells resulted in the generation of 3-chlorotyrosine. Moreover, activation of human neutrophils adherent to a L-tyrosine coated glass surface also stimulated 3-chlorotyrosine formation. Thus, in two independent models of phagocytosis human neutrophils convert L-tyrosine to 3-chlorotyrosine, indicating that a Cl2-like oxidant is generated in the phagolysosome. In both models, synthesis of 3-chlorotyrosine was inhibited by heme poisons and the peroxide scavenger catalase, implicating the myeloperoxidase-hydrogen peroxide system in the reaction. Collectively, these results demonstrate that myeloperoxidase generates Cl2 and that human neutrophils use an oxidant with characteristics identical to those of Cl2 during phagocytosis. Moreover, our observations suggest that phagocytes exploit the chlorinating properties of Cl2 to execute oxidative and cytotoxic reactions at sites of inflammation and vascular disease.

Chlorine↗

PubMed1996

Human phagocytes employ the myeloperoxidase-hydrogen peroxide system to synthesize dityrosine, trityrosine, pulcherosine, and isodityrosine by a tyrosyl radical-dependent pathway.

Myeloperoxidase, a heme protein secreted by activated phagocytes, may be a catalyst for lipoprotein oxidation in vivo. Active myeloperoxidase is a component of human atherosclerotic lesions, and atherosclerotic tissue exhibits selective enrichment of protein dityrosine cross-links, a well characterized product of myeloperoxidase. Tyrosylation of lipoproteins with peroxidase-generated tyrosyl radical generates multiple protein-bound tyrosine oxidation products in addition to dityrosine. The structural characterization of these products would thus serve as an important step in determining the role of myeloperoxidase in lipoprotein oxidation in the artery wall. We now report the identification and characterization of four distinct tyrosyl radical addition products generated by human phagocytes. Activated neutrophils synthesized three major fluorescent products from -tyrosine; on reverse phase HPLC, each compound coeluted with fluorescent oxidation products formed by myeloperoxidase. We purified the oxidation products to apparent homogeneity by cation and anion exchange chromatographies and identified the compounds as dityrosine (3,3'-dityrosine), trityrosine (3,3',5',3"-trityrosine) and pulcherosine (5-[4"-(2-carboxy-2-aminoethyl)phenoxy]3, 3'-dityrosine) by high resolution NMR spectroscopy and mass spectrometry. Additionally, we have found that dityrosine is a precursor to trityrosine, but not pulcherosine. In a search for a precursor to pulcherosine, we identified isodityrosine (3-[4'-(2-carboxy-2-aminoethyl)phenoxy]tyrosine), a non-fluorescent product of L-tyrosine oxidation by human phagocytes. Our results represent the first identification of this family of tyrosyl radical addition products in a mammalian system. Moreover, these compounds may serve as markers specific for tyrosyl radical-mediated oxidative damage in atherosclerosis and other inflammatory conditions.

Free Radicals↗

PubMed1996

Isotope dilution mass spectrometric measurements indicate that arachidonylethanolamide, the proposed endogenous ligand of the cannabinoid receptor, accumulates in rat brain tissue post mortem but is contained at low levels in or is absent from fresh tissue.

Arachidonylethanolamide (AEA) isolated from porcine brain binds to cannabinoid receptors, mimics cannabinoid pharmacologic effects, and has been proposed as an endogenous cannabinoid receptor ligand. Demonstration of co-distribution of AEA and cannabinoid receptors in various brain regions could provide supportive evidence for this role. We have performed isotope dilution mass spectrometric measurements of AEA and have demonstrated AEA production by rat tissue homogenates in vitro from exogenous arachidonate and ethanolamine. No detectable endogenous AEA (<3.5 pmol/g of tissue) was observed in fresh rat brain, whether or not inhibitors of AEA hydrolysis were present during tissue processing. AEA (>1 nmol/g) was produced during saponification of brain phospholipid extracts. This appears not to reflect hydrolysis of N-arachidonylethanolamine phospholipid precursors of AEA, because Streptomyces chromfucsis phospholipase D, which is active against NAPE, failed to generate AEA from brain phospholipids despite substantial conversion of phospholipids to phosphatidic acid. Such experiments suggested that the abundance of N-arachidonylethanolamine phospholipid in fresh rat brain may be less than 1 in 10(6) phospholipid molecules. AEA generated during saponification of tissue phospholipids appears to arise from base-catalyzed aminolysis of arachidonate-containing glycerolipids, because AEA was produced from synthetic (1-stearoyl, 2-arachidonoyl)-phosphatidylethanolamine under saponification conditions, and the amount produced increased 300-fold when free ethanolamine was included in the hydrolysis solution. Although AEA was not detectable (<0.17 pmol/mg of protein) in fresh rat brain, AEA accumulated post mortem to levels of 126 pmol/mg of brain protein. These findings do not exclude the possibility that AEA is rapidly synthesized and degraded locally in vivo, but they indicate that the AEA content of fresh rat brain and of NAPE precursors from which AEA might be derived are exceedingly low and that AEA can be produced artifactually from biological materials.

Animals↗

PubMed1996

Determination of benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine in human urine by capillary gas chromatography/negative ion chemical ionization mass spectrometry.

We report an isotope dilution mass spectrometry method using capillary gas chromatography/negative ion chemical ionization to quantitate urine concentrations of benzidine (BZ) and of its acetylated metabolites N-acetylbenzidine (ABZ) and N,N'-diacetylbenzidine (DABZ). Urine samples were purified by solid-phase extraction columns, reduced with LiAlH4/THF, and derivatized with pentafluoropropionic anhydride. The derivatives were measured by selected ion monitoring relative to deuterium-labeled internal standards. A detection limit as low as 0.5, 0.8, and 1.5 ppt for BZ, ABZ, and DABZ, respectively, can easily be achieved. Urinary concentrations of ABZ substantially exceed those of either BZ or of DABZ in workers exposed to BZ or BZ-based dyes. This method has been successfully used to measure BZ, ABZ, and DABZ in 1.0-ml urine samples collected from workers involved in manufacturing BZ and BZ-based dyes. The method should be applicable to the measurement of other aromatic amines and their acetylated metabolites.

Benzidines↗

PubMed1996

p-Hydroxyphenylacetaldehyde is the major product of L-tyrosine oxidation by activated human phagocytes. A chloride-dependent mechanism for the conversion of free amino acids into reactive aldehydes by myeloperoxidase.

Reactive aldehydes generated during lipid peroxidation have been implicated in the pathogenesis of atherosclerosis as well as other inflammatory diseases. A potential catalyst for such reactions is myeloperoxidase, a hemeprotein secreted by activated phagocytes. We now report that activated neutrophils utilize the myeloperoxidase-H2O2-chloride system to convert L-tyrosine to p-hydroxyphenylacetaldehyde. Production of p-hydroxyphenylacetaldehyde was nearly quantitative at physiological concentrations of L-tyrosine and chloride. Aldehyde generation required myeloperoxidase, H2O2, L-tyrosine, and chloride ion; it was inhibited by the H2O2 scavenger catalase and by the heme poisons azide and cyanide. Phorbol ester- and calcium ionophore-stimulated human neutrophils likewise generated p-hydroxyphenylacetaldehyde from L-tyrosine by a pathway inhibited by azide, cyanide, and catalase. Aldehyde production accounted for 75% of H2O2 generated by optimally stimulated neutrophils at plasma concentrations of L-tyrosine and chloride. Collectively, these results indicate that activated phagocytes, under physiological conditions, utilize myeloperoxidase to execute the chloride-dependent conversion of L-tyrosine to the lipid-soluble aldehyde, p-hydroxyphenylacetaldehyde, in near quantitative yield. Moreover, like aldehydes derived from lipid peroxidation, amino acid-derived aldehydes may exert potent biological effects in vascular lesions and other sites of inflammation.

Acetaldehyde↗

PubMed1996

Quantification of cholesterol tracers by gas chromatography--negative ion chemical ionization mass spectrometry.

Because of its high sensitivity, gas chromatography negative ion chemical ionization mass spectrometry (GC-NCI-MS) is a potentially valuable analytical tool for the study of cholesterol metabolism. Of several derivatives prepared for potential use in tracer studies pentafluorobenzoyl cholesterol was selected because it formed rapidly at ambient temperature and was stable for long periods, could be detected at a level of 1 fmol, and yielded a mass spectrum in which the molecular ion was the principal component. Hexadeuterated cholesterol tracer ([26,26,26,27,27,27-2H6]cholesterol) could be detected in dilutions up to 2700 in unlabeled cholesterol by selected ion monitoring with a coefficient of variation averaging 3.2%. In seven normal subjects tracer cholesterol was infused intravenously and plasma cholesterol enrichment was determined after 4 h. The measured rapidly miscible cholesterol pool was 391.0 +/- 38.6 mg cholesterol/kg. Negative ion mass spectrometry of pentafluorobenzyol cholesterol will facilitate analysis of both small amounts of natural cholesterol and labeled cholesterol in applications where sensitivity is critical.

Benzoates↗

PubMed1996

NADPH-dependent oxidation of benzidine by rat liver.

This study used liver microsomes from control an naphthoflavone-treated rats to evaluate NADPH-dependent oxidation of benzidine. With microsomes from beta-naphthoflavone-treated rats, the rates of formation of aqueous soluble metabolite (HPLC analysis) and protein and DNA binding were 835 +/- 81, 14.5 +/- 1.8 and 0.71 +/- 0.14 pmol/mg/min respectively. beta-Naphthoflavone treatment elicited 12.3-, 1.8- and 14.2-fold increases in benzidine metabolism compared with controls as judged by HPLC and protein and DNA binding respectively. For microsomes from treated animals, Km and Vmax values were 47 +/- 6 micromol and 1.13 +/- 0.16 nmol/mg protein/min respectively. All of the metabolic parameters were inhibited to varying degrees by glutathione (1 or 10 mM), N-acetylmethionine (10 mM) and ascorbic acid (10 mM). Following glutathione addition, at least two new metabolite peaks were observed, representing -6% of the total radioactivity recovered by HPLC. Neither metabolite was 3-(glutathion-S-yl)benzidine. Cytochrome P450 inhibitors (10 micro) specific for different members of cytochrome gene families 1-3 indicated that benzidine was metabolized by cytochrome P450 1A1/1A2. Ellipticine and alpha-naphthoflavone, specific 1A1/1A2 inhibitors, elicited 50% inhibition at -0.2 and 0.5 micro respectively. Electron impact and negative ion chemical ionization mass spectro- metry identified the aqueous soluble metabolite as 3-hydroxybenzidine. The lability of 3-hydroxybenzidine observed at pH > 7.0 was prevented by ascorbic acid. Thus, cytochrome P450 1A1/1A2 NADPH-dependent metabolism of benzidine to 3-hydroxybenzidine was demonstrated.

Animals↗

PubMed1996

The glutathione S-transferase M1 (GSTM1) null genotype and benzidine-associated bladder cancer, urine mutagenicity, and exfoliated urothelial cell DNA adducts.

Multiple studies in the general population have suggested that subjects with the glutathione S-transferase M1 (GSTM1)-null genotype, who lack functional GSTM1, are at higher risk for bladder cancer. To evaluate the impact of the GSTM1-null genotype on bladder cancer caused by occupational exposure to benzidine and to determine its influence on benzidine metabolism, we carried out three complementary investigations: a case-control study of bladder cancer among workers previously exposed to benzidine in China, a cross-sectional study of urothelial cell DNA adducts and urinary mutagenicity in workers currently exposed to benzidine in India, and a laboratory study of the ability of human GSTM1 to conjugate benzidine and its known metabolites in vitro. There was no overall increase in bladder cancer risk for the GSTM1-null genotype among 38 bladder cancer cases and 43 controls (odds ratio, 1.0; 95% confidence interval, 0.4-2.7), although there was some indication that highly exposed workers with the GSTM1-null genotype were at greater risk of bladder cancer compared to similarly exposed workers without this allele. However, the GSTM1 genotype had no impact on urothelial cell DNA adduct and urinary mutagenicity levels in workers currently exposed to benzidine. Furthermore, human GSTM1 did not conjugate benzidine or its metabolites. These results led us to conclude that the GSTM1-null genotype does not have an impact on bladder cancer caused by benzidine, providing a contrast to its association with elevated bladder cancer risk in the general population.

Benzidines↗

PubMed1995

Glucuronidation of N-hydroxy metabolites of N-acetylbenzidine.

Glucuronidation of N-hydroxy arylamines is thought to be a necessary step in their initiation of bladder cancer. This was evaluated for the N-hydroxy metabolites of N-acetylbenzidine (ABZ). N'-Hydroxy-N-acetylbenzidine (N'-HA), N-hydroxy-N-acetylbenzidine (N-HA) and N-hydroxy- N,N'-diacetylbenzidine (N-HDA) were synthesized. Except for N'-HA, these compounds were quite stable. Ascorbic acid and/or acidic pH increased the stability of N'-HA. When each N-hydroxy compound was added to reaction mixtures containing [14C]UDP-glucuronic acid, 3 mM ascorbic acid and human liver microsomes a new product was detected by HPLC. Emulgen 911 was a better detergent than Triton X-100 for expressing microsomal activity, with maximal glucuronidation observed with 0.3% Emulgen 911. At 0.125 mM amine the rate of glucuronidation was N-HDA >> N'-HA = benzidine > ABZ > N-HA. In contrast, at 0.5 mM amine the rate of glucuronidation of N-HA was only exceeded by N-HDA. At pH 5.5 and 37 degrees C the t1/2 for the enzymatically prepared glucuronide conjugates of ABZ, N'-HA and N-HA were 7.5 min and 3.5 and 1.8 h respectively. For N-HDA > 90% of this glucuronide remained after 24 h. At pH 7.4 and 37 degrees C the t1/2 for the glucuronide conjugates of ABZ and N-HA were 2.3 and 2 h respectively, with the amounts remaining after 24 h for N'-HA and N-HDA being 75 and 90% respectively. At pH 6.5 the t1/2 for N'-HA was 14 h. Thus only glucuronides of ABZ and N'-HA exhibit pH-dependent changes in t1/2. Compared with ABZ, glucuronides the N-hydroxy metabolites are more stable at acidic pH. Acidic urine would be more likely to hydrolyze the glucuronide conjugate of ABZ than those of its N-hydroxy metabolites. Because these results are different from that hypothesized for arylmonoamines, a new model was developed to explain the role of N-oxidation, N-glucuronidation and N-acetylation in the carcinogenesis of benzidine, an aryldiamine.

Acetylation↗

PubMed1994

Characterization of peroxidative oxidation products of dopamine by mass spectrometry.

We characterized three cytotoxic products, namely dopaminochrome (2,3-dihydro-1H-indole-5,6-dione), 2-(3,4-dihydroxyphenyl)-1-nitroethane and 2-(3,4,6-trihydroxyphenyl)-1-nitroethane. The compounds were separated from the incubation of dopamine (3,4-dihydroxyphenethylamine) with horseradish peroxidase which mimics the peroxidative activity of Prostaglandin H synthase. Incubation of 2-(3,4,6-trihydroxyphenyl)-1-nitroethane with NADPH-cytochrome c reductase led to the formation of 6-hydroxydopamine, a known neurotoxin. Several adducts were also isolated in this study. Oxidation of dopamine in the presence of N-acetylcysteine yielded a thioether conjugate namely, 5-S-(N-acetylcysteinyl)-3,4-dihydroxyphenethylamine. Reaction of the partially purified dopaminochrome with N-acetylcysteine permitted the isolation of another thioether conjugate which was tentatively identified as 7-S-(N-acetylcysteinyl)-5,6-dihydroxyindole. We also isolated the one-to-one condensation products of malonaldehyde with dopamine, norepinephrine and serotonin. The identities of these products were established by chemical synthesis and various mass spectrometric techniques.

Acetylcysteine↗

PubMed1994

Determination of cortisol in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry.

We report an isotope dilution mass spectrometry method using capillary gas chromatography negative ion chemical ionization for the determination of cortisol in human plasma. Cortisol was derivatized with pentafluoropropionic anhydride at room temperature and measured by selected ion monitoring of the molecular ion. This method utilized d4-cortisol as an internal standard and required only a simple correction for the isotope contribution. A sensitivity as low as 0.02 pmol (s/n > 10) can be achieved easily. Therefore a plasma sample as small as 0.1 ml can be used for the accurate measurement of cortisol in humans.

Acetonitriles↗