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.

Inhibition of feeding by a nitric oxide synthase inhibitor: effects of aging.

Nitric oxide has been demonstrated to play a role in the modulation of food intake. With advancing age, there is a physiological decrease in food intake. The effect of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on food intake in C57BL/6Nnia mice aged 3, 12 and 24 months was studied. L-NAME was more effective at decreasing food intake in 12- and 24-month-old mice than in the 3-month-old mice. NO synthase levels in the hypothalamus were increased in 16- and 25-month-old mice compared to 6-month-old mice (P < 0.01). NO synthase mRNA increased in 16- compared to 6-month-old mice, but decreased in 25-month-old mice. Overall, these studies may suggest that nitric oxide may play an increasingly important role in the feeding drive with advancing age.

Nitric oxide synthase levels in obese Zucker rats.

Nitric oxide has been demonstrated to play a role in the modulation of food intake. The Zucker fatty rat is an autosomal recessive genetic model of obesity. We measured nitric oxide synthase (NOS) in the hypothalamus and fundus of the stomach in Zucker (fa/fa) rats and their lean littermate controls (fa/?). NOS activity was decreased in both the hypothalamus and the fundus of the Zucker (fa/fa) rats compared to the littermate controls.

A new perspective on the inhibitory role of nitric oxide in sympathetic neurotransmission.

By using, as a model of sympathetic neurons, immortalized rat pheochromocytoma (PC12) cells differentiated by nerve growth factor (NGF), the effect of nitric oxide on sympathetic neurotransmission was examined. The NO donor sodium nitroprusside (SNP; 10(-4)-3 x 10(-4) M) caused an apparent inhibition of dopamine release from PC12 cells, as measured by HPLC. Studies, in the absence of cells, involving the incubation of dopamine (20 ng/ml) or norepinephrine (15 ng/ml) with SNP (10(-6)-3 x 10(-4) M) or authentic NO (6 x 10(-6)-3 x 10(-5) M) revealed a similar reduction in the detection of the catecholamines. In addition, absorption spectroscopy studies showed dopamine and norepinephrine to be oxidized by NO resulting in the formation of their respective quinone products. These observations, coupled with the finding that the ability of dopamine to raise cAMP levels within PC12 cells was reduced after incubation with SNP, reveal that NO inhibits the biological activity rather than the release of catecholamines.

An endogenous dopaminergic neurotoxin: implication for Parkinson's disease.

Oxidation of dopamine by monoamine oxidase results in the endogenous metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL). The toxicity of DOPAL for dopaminergic neurons was investigated using rat neostriatal synaptosomes, PC-12 cells and cultures of fetal rat dissociated mesencephalon. The Na(+)-dependent uptake of [3H]DOPAL in synaptosomes was inhibited by mazindol. DOPAL selectively inhibited dopamine uptake but not [14C]GABA uptake, induced membrane damage and liberation of dopamine into the medium. Incubation of PC-12 cells with 6.5 microM of DOPAL for 24 h caused degeneration of the neuritic process, and the number of viable cells were reduced by 25% of control. There were practically no surviving cells after 24 h of incubation with 33 microM of DOPAL. After 8 h of treatment with 33 microM of DOPAL, dopamine and 3,4-dihydroxyphenylacetic acid content in the cells were reduced by 38% and 53% of control. DOPAL-induced cell damage released lactic acid dehydrogenase into the incubation media. This toxic effect of DOPAL was time- and concentration-dependent. In mesencephalic cultures, after exposure to 33 microM of DOPAL, the surviving TH+ cells showed rounded cell body, and fibre network was highly reduced. These results indicate DOPAL is a neurotoxin and may be involved in the degeneration of dopaminergic neurons.

Prostaglandin H synthetase-mediated metabolism of dopamine: implication for Parkinson's disease.

Differences in prostaglandin H synthetase (PHS) activity in the substantia nigra of age- and postmortem interval-matched parkinsonian, Alzheimer's, and normal control brain tissue were assessed. Prostaglandin E2 (PGE2, an index of PHS activity) was higher in substantia nigra of parkinsonian brain tissue than Alzheimer's or control tissue. Incubation of substantia nigra slices with arachidonic acid (AA) increased PGE2 synthesis. Dopamine stimulated PHS synthesis of PGE2. [3H]Dopamine was activated by PHS to electrophilic intermediate(s) that covalently bound to DNA, microtubulin protein, bovine serum albumin, and sulfhydryl reagents. When AA was replaced by hydrogen peroxide, PHS/H2O2-supported binding proceeded at rates similar to those observed with PHS/AA. Indomethacin and aspirin inhibited AA-mediated cooxidation of dopamine but not H2O2-mediated metabolism. PHS-mediated metabolism of dopamine was not affected by monoamine oxidase inhibitors. Substrate requirements and effects of specific inhibitors suggest cooxidation of dopamine is mediated by the hydroperoxidase activity of PHS. 32P-postlabeling was used to detect dopamine-DNA adducts. PHS/AA activation of dopamine in the presence of DNA resulted in the formation of five dopamine-DNA adducts, i.e., 23, 43, 114, 70, and 270 amol/micrograms DNA. DNA adduct formation was PHS, AA, and dopamine dependent. PHS catalyzed cooxidation of dopamine in dopaminergic neuronal degeneration is discussed.

Measurement of nitric oxide synthase and its mRNA in genetically obese (ob/ob) mice.

Recent studies have suggested a role for nitric oxide (NO) in the regulation of food intake. The obese (ob/ob) mouse is a genetic model of obesity. Previously, it has been demonstrated that ob/ob mice show a marked weight reduction when treated with a nitric oxide synthase inhibitor. In the studies reported here, we demonstrate increased levels of nitric oxide synthase (NOS) and its mRNA in the hypothalamus of genetically obese (ob/ob) mice compared to their lean littermate controls (ob/c). NOS levels were 0.016 +/- 0.001 nmol/mg/min in ob/ob compared to 0.009 +/- 0.001 in ob/c (p < 0.01) and NOS mRNA was 32.0 +/- 5.0 pg NOS mRNA/mg total RNA in ob/ob compared to 12.4 +/- 4.0 in ob/c (p < 0.05). These studies further support the possibility of a role for nitric oxide in the regulation of food intake.

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.

Phenylbutazone peroxidatic metabolism and conjugation.

Phenylbutazone, a nonsteroidal anti-inflammatory drug, elicits therapeutic as well as toxic effects by unknown pathways. Phenylbutazone was shown to form a conjugate with the heterocyclic amine bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT). To understand further the reactivity of these compounds, this study was conducted to identify the conjugate formed and determine the mechanism of conjugate formation. Both prostaglandin H synthase and horseradish peroxidase catalyzed conjugate formation. This conjugate was identified by 1H-NMR to be 4-[2-amino-4-(5-nitro-2-furyl)-5-thiazolyl]-4-butyl-1,2-diphenyl-3,5- pyrazolidinedione. Phenylbutazone-mediated oxygen uptake was inhibited by ANFT (0.1 mM) and the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (200 mM) and tert-nitrosobutane (4 mM). By contrast, phenol (0.005 to 0.25 mM) and aminopyrine (0.4 mM) stimulated oxygen uptake. None of these agents mediated oxygen uptake in the absence of phenylbutazone. Conjugate formation was significantly increased by phenol (0.005-0.25 mM) and aminopyrine (0.4 mM), as well as in the absence of oxygen. Conjugate formation was inhibited by 5,5-dimethyl-1-pyrroline-N-oxide (200 mM), tert-nitrosobutane (4 mM), ascorbic acid (2 mM), and 95% oxygen. Horseradish peroxidase initiated conjugate formation at much lower concentrations than it metabolized ANFT. The stoichiometric relationship between phenylbutazone and ANFT, with respect to conjugate formation, was complex. With the concentration of ANFT fixed at 0.05 mM, phenylbutazone exhibited saturation kinetics with a Km of 0.2 mM. In contrast, saturation kinetics were not observed with ANFT.Km values for ANFT varied with the concentration of phenylbutazone used.(ABSTRACT TRUNCATED AT 250 WORDS)

Confirmation of a dopamine metabolite in parkinsonian brain tissue by gas chromatography-mass spectrometry.

Gas chromatography-mass spectrometry was used to identify a dopamine metabolite isolated from the substantia nigra of parkinsonian brain tissue. Incubation of dopamine with monoamine oxidase B gave the same product which was identified as 3,4-dihydroxyphenylacetaldehyde. The structure of the compound was established by chemical synthesis, metastable ion measurement and high-resolution mass spectrometry.

Effects of testosterone replacement therapy in old hypogonadal males: a preliminary study.

OBJECTIVE: To examine the effects of testosterone administration to older hypogonadal males (bioavailable testosterone less than 70 ng/dL). DESIGN: Alternate-case controlled trial. SETTING: St. Louis University. PATIENTS: Eight males (mean age 77.6 +/- 2.3 years) who received testosterone and six males (mean age 76 +/- 2.3 years) who served as controls. Selected from alumni of the SHEP trial and attendees at the St. Louis University Impotence Clinic. INTERVENTIONS: Testosterone enanthate (200 mg/mL) was administered intramuscularly to the treatment group every 2 weeks for 3 months. MEASUREMENTS: Serum testosterone, bioavailable testosterone and estradiol, weight, % body fat, right hand muscle strength, balance, cholesterol, HDL-cholesterol, hematocrit, BUN, creatinine, albumin, calcium, PTH, 25(OH) vitamin D, 1,25(OH)2 vitamin D, osteocalcin, prostate-specific antigen, and fructosamine. RESULTS: Males who received testosterone had a significant increase in testosterone and bioavailable testosterone concentration, hematocrit, right hand muscle strength and osteocalcin concentration. They had a decrease in cholesterol (without a change in HDL-cholesterol) levels and decreased BUN/Creatinine ratios. CONCLUSION: These preliminary findings support the need for long term studies of testosterone therapy in older hypogonadal males.

Metabolism and disposition of bladder carcinogens in rat and guinea pig: possible mechanism of guinea pig resistance to bladder cancer.

The metabolism and disposition of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) were studied in rat and guinea pig. Rat is susceptible whereas guinea pig is resistant to FANFT-induced bladder cancer. Rats and guinea pigs were p.o. administered either 2-[14C]ANFT or 2-[14C]FANFT (100 mg/kg), and 18-h urine and feces were collected. Tissue distribution of radiolabel was determined. In both species, the highest concentrations of radioactivity expressed as nmol/g tissue were observed in the urine and intestines. Urinary metabolites were separated by high-performance liquid chromatography and radioactivity determined by radioanalytical detection. FANFT was not detected in urine from either species under any experimental condition. More ANFT was observed in urine following FANFT than ANFT administration. This deformylation-dependent excretion of FANFT was demonstrated in both species and has been previously described as renal metabolic/excretory coupling. Less ANFT, the carcinogen more proximate than FANFT, is excreted in guinea pigs compared with rats. A unique ANFT metabolite was identified in guinea pig but not rat urine. This metabolite represented 80 and 18% of radioactivity recovered in guinea pig urine following ANFT and FANFT administration, respectively. A metabolite produced by guinea pig liver and kidney microsomes in the presence of uridine-5'-diphosphoglucuronic acid coeluted with this unique metabolite. The urinary metabolite was characterized using hydrolytic enzymes, acid hydrolysis, and mass spectrometry and identified as an ANFT-N-glucuronide. A unique UDP-glucuronosyl-transferase appears to be responsible, at least in part, for the reduced amount of free ANFT excreted by guinea pigs compared with rats. Reduced levels of urinary ANFT observed in guinea pigs may partially explain the resistance of this species to FANFT-induced bladder cancer.

Mechanism of peroxidative activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT): comparison with benzidine.

The mechanism of activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) was investigated by comparison with benzidine. In comparison with benzidine, ANFT has a higher electrochemical potential (approximately 700 mV) and is less effective as a reducing co-substrate for either prostaglandin H synthase (PHS) or horseradish peroxidase. Activation was monitored by measuring binding to protein (BSA) and DNA. ANFT binding to protein was reduced by indomethacin, a fatty acid cyclooxygenase inhibitor; phenol and aminopyrine, competitive reducing co-substrates; ascorbic acid, an antioxidant; and glutathione, thioether conjugate formation. These results are consistent with those previously reported for benzidine and demonstrate a peroxide co-substrate requirement, interaction of peroxidase with amine, formation of reactive intermediates and inactivation of reactive intermediates. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), a radical trap, also reduced ANFT binding to protein. Similar results were observed whether activation by PHS or horseradish peroxidase was investigated. Peroxidative activation of ANFT and benzidine to bind DNA was inhibited by these test agents in a manner similar to that observed with protein except that DMPO did not reduce binding. In addition, 2-methyl-2-nitrosopropane and methyl viologen, which are radical traps, and methionine and p-nitrobenzyl-pyridine, which are strong nucleophiles, did not reduce ANFT or benzidine binding to DNA. These agents also did not prevent binding of benzidinediimine, the two-electron product of benzidine oxidation, to polydeoxyguanosine. Glutathione inhibited diimine binding by forming a conjugate. Results demonstrate that activation of ANFT to bind protein and DNA is similar to benzidine. Peroxidative activation of benzidine occurs by both one- and two-electron oxidation. A similar mechanism would explain ANFT binding to protein (one electron) and DNA (two electron).

Thermospray high performance liquid chromatography/mass spectrometric identification of a bladder carcinogen metabolite isolated from guinea pig urine.

An in vivo urinary metabolite of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl) thiazole was isolated from guinea pig urine and was identified by direct analysis using thermospray mass spectrometry/high-performance liquid chromatography as 1-(2-amino-4-(5-nitro-2-furyl)-2-thiazolyl)-1-deoxy-beta-D-glucopyran uronic acid. The structure of this metabolite was also established by chemical synthesis. Both positive and negative ion thermospray mass spectrometry of the conjugate showed fragment ions resulting from cleavage across the pyran ring of the glucuronic acid comprising of aglycone moiety. These characteristic fragment ions may be diagnostic for identification of N-glucuronides from O-glucuronides.

Metabolism and disposition of benzidine in the dog.

The dog is an animal model for assessing aromatic amine-induced bladder cancer. For this reason, metabolism and disposition of benzidine in dog was assessed. Dogs were administered a 1 mg/kg i.v. dose of [3H]benzidine (16.4 mCi/mmol). The plasma t1/2 of the radiolabeled material (benzidine plus metabolites) was significantly longer (approximately 3 h) than authentic benzidine (less than 30 min). During the 5 h experiment, the majority of radiolabel was associated with bile, urine and carcass. Bladder transitional epithelium exhibited a consistently higher concentration of bound radioactivity than bladder muscle. A significant amount of binding was observed in DNA from liver, kidney and bladder. DNA from bladder transitional epithelium exhibited the highest concentration of radioactivity. Approximately 30% of the radioactivity recovered following HPLC of urine or bile was identified as unmetabolized benzidine. 3-Hydroxybenzidine was a major metabolite identified in bile (8%) but not urine. Urine samples treated with acid, base or sulfatase yielded 3-hydroxybenzidine (6%) as a major hydrolysis product. Similar treatment of bile samples did not result in increased amounts of 3-hydroxybenzidine. Neither N-acetylated nor N-methylated metabolites of benzidine were observed in urine or bile. Thus, considerable metabolism of benzidine occurs in dogs by pathways that are yet to be determined.

Metabolism of aromatic and heterocyclic amine bladder carcinogens: bioanalytical considerations.

Aromatic and heterocyclic amines are environmental chemicals which can cause bladder cancer in man. Because these chemicals cause carcinomas at a site distal to their portals of entry, metabolic processes are involved in initiation of their carcinogenic effects. N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and its deformylated analogue, ANFT, were used as model compounds to assess metabolism. Electrochemical properties of ANFT made liquid chromatography with electrochemical detection a specific and sensitive method for analysis. Peroxidatic metabolism of ANFT by prostaglandin H synthase (PHS) in the presence of N-acetylcysteine resulted in the formation of 2-amino-4-(5-nitro-2-furyl)-5-(N-acetylcystein-S-yl)thiazole (ANFT-MA). This thioether product has an oxidation potential significantly lower than ANFT. Rat urinary excretion of ANFT-MA was significantly decreased with peroxidase inhibitors, 6-n-propyl-2-thiouracil and methimazole. Inhibitors did not alter excretion of ANFT or prostaglandin E2, a PHS product of arachidonic acid metabolism. 1H and 13C-NMR were selected to explore potential structural differences between ANFT and FANFT which might explain preferential PHS metabolism of ANFT. Evidence for a "zwitterion" configuration for ANFT but not FANFT was observed. ANFT in the "zwitterion" configuration would be a better reducing co-substrate. Chemical synthesis and GC-MS fragmentation patterns identified 3-(2,3-dihydro-1-methyl-2-pyrrolyl)pyridine as a peroxidatic product of nicotine metabolism. This peroxidatic product was found in urine from a cigarette smoker in an amount approximately 6% that observed for continine. Thus, a potential rôle for peroxidative metabolism was demonstrated in man.

Detection of 3,4-dihydroxyphenylglycolaldehyde in human brain by high-performance liquid chromatography.

The monoamine oxidase A metabolite of noradrenaline, 3,4-dihydroxyphenylglycolaldehyde, is the precursor of 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol, metabolites of noradrenaline. Owing to difficulties in purifying this aldehyde, it has not been previously characterized or identified in biological sources. This paper describes an enzymatic synthesis, purification, and characterization of 3,4-dihydroxyphenylglycolaldehyde. The aldehyde metabolite is identified in postmortem human brain using high-performance liquid chromatography and electrochemical detection. We estimate the concentration in human hippocampus to be 0.164 +/- 0.05 nmol/g. The importance of this aldehyde metabolite of noradrenaline is discussed.

Renal metabolism of formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]-hydrazide.

Formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]-hydrazide (FNT) is a potent renal carcinogen in the rat. This study assessed the metabolism of FNT by the isolated perfused rat kidney and whole rat. The glomerular filtration rate and the fractional excretion of sodium for the isolated perfused kidney indicated that under the conditions of these experiments FNT did not alter these renal parameters. The half-life (t1/2) for FNT in the isolated perfused kidney was 67 +/- 8 min. Using HPLC, a metabolite of FNT was observed in urine from the isolated perfused kidney. This metabolite had absorbance at 385 nm but not 254 nm and could not be detected electrochemically at +500 mV. While the excretion of FNT decreased with time of perfusion, the metabolite excretion increased. Whole animal studies demonstrated that FNT is rapidly cleared from blood within the first 5 min of administration. The FNT metabolite was excreted at approximately the same rate from 0-30 and 30-60 min after FNT administration. The metabolite was not observed in media from FNT perfused kidneys or plasma from animals administered FNT. Analysis of purified metabolite by liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS) determined the structure to be 5-nitro-2-furonitrile. This structure assignment was verified by chemical synthesis. Results demonstrate target organ metabolism of carcinogen.

'Revised' mass spectral identification of 2-amino-4-(5-nitro-2-furyl)thiazole metabolites.

The electron ionization mass spectra of 2-amino-4-(5-nitro-2-furyl)thiazole metabolites obtained from microsomal incubations and chemical syntheses were studied. The identities of the metabolites were established by chemical ionization, high resolution, and metastable measurements. The compounds studied showed multiple modes of cleavage, skeletal rearrangements and hydrogen back-transfer.

Lung prostaglandin H synthase and mixed-function oxidase metabolism of nicotine.

Nicotine, a major constituent of cigarette smoke, was metabolized by lung microsomes to an aqueous soluble metabolite after addition of arachidonic acid. Similar results were observed with ram seminal vesicle microsomes. Metabolism was inhibited by indomethacin, propylthiouracil and methimazole but not glutathione. Data are consistent with metabolism being catalyzed by the hydroperoxidase activity of prostaglandin H synthase. The product was identified by mass spectrometry as 3-(2,3-dihydro-1-methyl-2-pyrrolyl)pyridine. Addition of NADPH resulted in formation of a different aqueous soluble product and also an organic extractable product. NADPH-dependent products were inhibited by 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine, suggesting mixed-function oxidase catalyzed metabolism. The organic soluble product was identified as cotinine. Cotinine formation was inhibited by glutathione. 3-(2,3-dihydro-1-methyl-2-pyrrolyl)Pyridine was identified in urine from rabbits administered nicotine and from a male cigarette smoker. The amount of peroxidatic product in urine from rabbit and humans was 15 and 6%, respectively, that observed for cotinine. Thus, peroxidation represents a new metabolic pathway for nicotine which involves the peroxidatic activity of prostaglandin H synthase.