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PubMed1984

Demonstration of microsomal oxygenation of the benzo ring of 6-nitrobenzo[a]pyrene by thin-layer chromatography.

To explain the biological activity of 6-nitrobenzo[a]pyrene (6-nitroBaP), male Sprague-Dawley rats were induced with 3-methylcholanthrene. Liver microsomes were incubated with magnesium chloride, an NADPH generating system and 6-nitroBaP in acetone. The mixture was chilled under oxygen-free argon gas and protein was precipitated with an equal volume of cold methanol containing triethylamine. Protein was further precipitated with zinc and sodium sulfate and centrifuged. Both the sediment and the supernatant were extracted with benzene and ethyl acetate. The organic extract was washed with water, 2% sodium hydroxide solution, water and then dried with anhydrous sodium sulfate. Solvents were removed and the residue was chromatographed on silica gel plates with hexane containing increasing amounts of benzene. The UV and mass spectra of products were examined. Liver microsomal metabolites of 6-nitroBaP consisted of 7,8- and 9,10-dihydrodiols and also benzo[a]pyrene (BaP) and BaP-quinones. cis-Forms of 6-nitroBaP-7,8- and -9,10-dihydrodiols were synthesized.

Animals↗

PubMed1988

Chromatographic and spectral investigations on the in vivo metabolites of 6-nitrobenzo[a]pyrene.

6-Nitrobenzo[a]pyrene (6-NBaP) occurs in the environment, is mutagenic in the Ames assay in the presence of added S9 and is carcinogenic to male but not female mouse liver when injected intraperitoneally (i.p.) into mice. In order to understand what kinds of active metabolites could have been produced in vivo, both male and female mice were injected i.p. with 6-NBaP in dimethyl sulfoxide. Twenty-four hours after injection, urine, feces, blood, liver and spleen (non-target tissue) were examined for metabolites by chromatographic and high-resolution mass spectral means. On the basis of the mass spectral fragmentation patterns of synthetic and metabolic standards, it was observed that both male and female animals excreted ring-hydroxylated metabolites of 6-NBaP in the urine to differing extents. Male animals additionally excreted 6-aminobenzo[a]pyrene and the significance of this observation is discussed.

Aging↗

PubMed1997

Determination of glutathione, cysteine and N-acetylcysteine in rabbit eye tissues using high-performance liquid chromatography and post-column derivatization with 5,5'-dithiobis(2-nitrobenzoic acid).

A high-performance liquid chromatographic method to determine glutathione, cysteine and N-acetylcysteine in rabbit retina, vitreous and lens has been developed. The thiols are separated using a 25 x 0.46-cm octadecylsilane column with 0.5 M phosphate buffer, pH 3, as mobile phase. The detection, at 412 nm, involves a post-column derivatization with 5,5-dithiobis(2-nitrobenzoic acid) in presence of cationic micelles of hexadecyltrimethylammonium bromide that enhances the sensitivity. The detection limits are 0.21, 0.92 and 0.61 mumol/g wet sample for glutathione, cysteine and N-acetylcysteine, respectively.

Acetylcysteine↗

PubMed1997

Metabolism of isomeric nitrobenzo[a]pyrenes leading to DNA adducts and mutagenesis.

We have been interested in determining the structural and electronic features that may be useful in predicting the mutagenic activity of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs). We have previously found that a correlation between structural and electronic features and direct-acting mutagenicity in Salmonella typhimurium cannot be made using nitro-PAHs with different molecular size. In this study, a series of structurally related nitro-PAHs, the environmental contaminants 1-, 3-, and 6-nitrobenzo[alpha]pyrene (NBaP) and their derivatives, was used to determine structure-activity relationships. It was found that isomeric NBaPs are activated to DNA damaging and mutagenic derivatives by nitroreduction, ring-oxidation, or by a combination of these two pathways. A general finding was that NBaPs and derivatives with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct-acting mutagenicity in S. typhimurium strains TA98 and TA100. In this paper, we also discuss the effect of the location of the nitro group on the metabolism and the mutagenicity of NBaPs and the effect of oxygen-containing functional groups on the mutagenicity of NBaP derivatives. These findings provide a useful molecular basis for interpreting and predicting the direct-acting mutagenicity of nitro-PAHs.

Animals↗

PubMed1998

Reactivation kinetics of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified creatine kinase reactivated by dithiothreitol.

The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified creatine by dithiothreitol has been studied using the kinetic theory of the substrate reaction during modification of enzyme activity as previously described by C.L. Tsou (Adv. Enzymol. Rel. Areas Mol. Biol. 61 (1988) 381-436). The results show that the modified creatine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analogue markedly slows the apparent reactivation rate constant, while creatine shows no effect. The substrates creatine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO-3 cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of creatine kinase are discussed. It is suggested that the cysteine may be located in the hinge area of the two domains of creatine kinase. The reactive cysteine of creatine kinase may play an important role not in the binding to the transition-state analogue but in the conformational changes caused by the transition-state analogue.

Adenosine Diphosphate↗

PubMed1999

Tumorigenicity and liver tumor ras-protooncogene mutations in CD-1 mice treated neonatally with 1- and 3-nitrobenzo[a]pyrene and their trans-7,8-dihydrodiol and aminobenzo[a]pyrene metabolites.

The environmental pollutants 1- and 3-nitrobenzo[a]pyrene (1- and 3-NBaP) are metabolized by mammalian microsomes through ring oxidation to 1-NBaP trans-7,8-dihydrodiol and 3-NBaP trans-7,8-dihydrodiol, and by nitroreduction to 1- and 3-aminobenzo[a]pyrene. To determine if these compounds are tumorigenic, 1- and 3-NBaP, along with several of their metabolites and the parent benzo[a]pyrene (BaP) and its trans-7,8-dihydrodiol metabolite, were tested in the neonatal CD-1 mouse bioassay. Male mice were administered i.p. injections at a total dose of 100 or 400 nmol per mouse on 1, 8 and 15 days after birth. While the liver tumor incidences for BaP, BaP trans-7,8-dihydrodiol, and the positive control 6-nitrochrysene (6-NC) were significantly higher than in the solvent control animals, all the other tested compounds exhibited no tumorigenicity. The frequency of Ha- and Ki-ras mutations in liver tumors of mice treated with BaP, BaP trans-7,8-dihydrodiol, and 6-NC were higher than in the few liver tumors isolated from control mice or mice treated with the NBaPs or their metabolites. Since 1- and 3-NBaP and their metabolites are potent mutagens in the Salmonella assay and moderate mutagens in the Chinese hamster ovary (CHO) mammalian mutagenicity assay, our results indicate that the in vitro mutagenicity of these compounds does not correlate with their tumorigenicity.

Adenoma, Liver Cell↗

PubMed1998

Comparative tumorigenicity of 1- and 3-nitrobenzo[a]pyrenes, and 3,6- and 1,6-dinitrobenzo[a]pyrenes in F344/DuCrj rats.

Our earlier study revealed that 1- and 3-nitrobenzo[a]pyrene (NBP), 1,6- and 3,6-dinitrobenzo[a]pyrene (DNBP), nitrated derivatives of benzo[a]BP (BP), are present in the environment. These derivatives are potent mutagens for Salmonella tester strains and we have preliminarily reported them to be carcinogenic in F344/DuCrj rats. In this study, the tumorigenic action of 1- and 3-NBP, 1.6- and 3,6-DNBP, and BP induced by subcutaneous injection into rats was found to differ according to the NO2-substitution in the BP structure. The chemicals were suspended in equal volumes of beeswax and tricaprylin, and rats were subcutaneously injected with single doses of 500, 1000, and 2000 microg for 1- and 3-NBP, and of 8, 40, 200, and 1000 microg for 3,6- and 1,6-DNBP, and BP as a positive control. 3,6-DNBP and BP induced tumors in a dose-dependent manner at the injection site. Rats given 1000 microg of 3,6-DNBP (2924 nmol) and BP (3968 nmol) developed subcutaneous tumors at the rate of 70 and 80%, respectively, and those given a minimum dose of 23 nmol for 3.6-DNBP and 32 nmol for BP per rat developed tumors at a rate of 4.8 and 18.2%, respectively. However, rats given 500 and 1000 microg of 1- and 3-NBP did not develop any tumors while those given a high dose, 2000 microg, of each chemical developed tumors at only one of ten animals used. It was concluded, therefore, that these chemicals are weak carcinogens. Histologically, most of the tumors were malignant fibrous histiocytomas. Rats given various doses of 1,6-DNBP did not develop any tumors at the injection site. The failure of 1,6-DNBP to induce tumors may involve its metabolites because of the lower mutagenicity of its reduction products, 1-nitroso-6-NBP and 1-amino-6-NBP. It is suggested, therefore, that tumorigenicities of NBPs and DNBPs differ according to the NO2-substitution on the chemical structure, which may be due to the possible nitroreduction of the chemicals.

Animals↗

PubMed2000

Analysis of 4-hydroxyproline using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol derivatization and micellar electrokinetic chromatography combined with laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection was used for internal standard (pyrrolidinol) based quantification of 4-hydroxyproline (Hyp) in muscle hydrolysates. Hydrolysates were first derivatized with o-phthaldialdehyde to reduce primary amine interference and then 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD) to enable selective detection of secondary amines. This method allows for rapid and sensitive detection of hydroxyproline in dilute hydrolysates using commercially available capillary electrophoresis equipment.

Amino Acids↗

PubMed1997

Development of a sensitive method for quantitation of ABT-089 in plasma using fluorescence labeling with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole.

A high-performance liquid chromatography method for the quantitation of ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine] (I), a new structural type of cholinergic channel modulators (ChCM), is described in this paper using 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescent-labeling reagent. The method combined an optimized liquid-liquid extraction from plasma followed by pre-column derivatization to yield a fluorescence product. The selectivity, sensitivity, and reproducibility of this method were found to be excellent. This method was applied to the determination of ng/ml plasma and tissue levels of ABT-089 and similar compounds in biological samples.

4-Chloro-7-nitrobenzofurazan↗

PubMed1997

HPLC determination of biologically active thiols using pre-column derivatisation with 5,5'-dithio-(bis-2-nitrobenzoic acid).

5,5'-Dithio-(bis-2-nitrobenzoic acid), Ellmans reagent (ESSE), is used as a pre-column derivatisation reagent for the determination of biologically active thiols by HPLC. D-penicillamine, N-acetyl-d-penicillamine, N-acetylcysteine, cysteine, captopril and thiomalic acid all give well resolved derivatives. The calibration graph and reproducibility (%R.S.D. +/- 1.3%) for the analysis of glutathione indicates that the method could be used for quantitative analysis. ESSE is widely used as a reagent in thiol determinations by electronic spectroscopy via the detection of the Ellmans anion (ES-) generated without any prior separation procedures. However, there are considerable reservations over its use for the spectrophotometric determination of thiols because of the possibility of side reactions which generate another Ellmans based species (ESO2-). The assay described determines the thiol as a derivatised mixed disulphide (ESSR) and since speciation between the anion ES- and the oxidation product ESO2- occurs it enables the process of oxidation to be monitored simultaneously.

Chromatography, High Pressure Liquid↗

PubMed1999

Determination of ABT-089 in human plasma by high performance liquid chromatography using in situ precolumn derivatization with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole.

ABT-089 is a potent, selective neuronal cholinergic channel modulator with cognition enhancing activity in several animal paradigms. A simple and sensitive chromatographic method for the specific determination of ABT-089 in human plasma has been developed and validated. The method utilizes in situ precolumn fluorescence derivatization of the sample with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) prior to liquid-liquid extraction followed by reverse phase HPLC and fluorescence detection (lambda(ex) 495 nm, lambda(em) 533 nm). The described method significantly simplifies sample preparation. The derivatized product was separated from interference using a YMC ODS-AQ, 5 microm, 250x4.6 mm i.d. column using a mobile phase consisting of 30:5:65 (v:v:v) acetonitrile/methanol/aqueous buffer at a flow rate of 0.75 ml min(-1). The aqueous buffer consisted of 0.01 M tetramethylammonium perchlorate, 0.1% (v:v) trifluoroacetic acid, pH 3.0. An Alltech Absorbosphere CN, 5 microm, cartridge guard column was also used before the analytical column. Plasma samples were alkalinized with 0.1 M NaHCO3, 300 microl of a 1 mg ml(-1) ethanolic solution of NBD-F was added and the samples were heated in a water bath for 10 min at 50 degrees C. The samples were then extracted with tert-butylmethylether, evaporated to dryness and then reconstituted in mobile phase. For 1 ml of plasma, a limit of quantitation (LOQ) of 1.6 ng ml(-1) was obtained. The method was linear from 1.6 to 836 ng ml(-1). Inter and intra-day assay RSD (n = 6) were less than 9%. Accuracy determinations showed the quality control samples to range between 88-114% of the theoretical concentration.

4-Chloro-7-nitrobenzofurazan↗

PubMed1990

Mechanism for activation of the 4-nitrobenzo-2-oxa-1,3-diazole-labeled sarcoplasmic reticulum ATPase by Ca2+ and its modulation by nucleotides.

The mechanism for activation of sarcoplasmic reticulum ATPase by Ca2+ was investigated in 2 mM MgCl2 and 0.1 M KCl at pH 6.5 and 11 degrees C by using enzyme preparations in which a specific amino acid residue (Cys-344) was labeled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) [Wakabayashi, S., Imagawa, T., & Shigekawa, M. (1990) J. Biochem. (Tokyo) 107, 563-571]. We compared the kinetics of binding and dissociation of Ca2+ from the enzyme with those of the accompanying NBD fluorescence changes. The fluorescence rise following addition of Ca2+ proceeded monoexponentially. At 2-100 microM Ca2+ and in the absence of nucleotides, the Ca2(+)-induced fluorescence rise and Ca2+ binding to the enzyme proceeded at similar rates, which were almost independent of the Ca2+ concentration. In contrast, the fluorescence decrease induced by Ca2+ removal was slower than the Ca2+ dissociation, and both of these processes were inhibited markedly by increasing medium Ca2+. ATP by binding at 1 mol/mol of the phosphorylation site markedly accelerated both the Ca2(+)-induced fluorescence rise and Ca2+ binding, ADP and AMPPNP but not GTP also being effective. In contrast, ADP minimally affected the NBD fluorescence decrease and the Ca2+ dissociation. These data are consistent with a reaction model in which binding of Ca2+ occurs after the conformational transition of the free enzyme from a state (E2) having low affinity for Ca2+ to one (E1) having high affinity for Ca2+ and in which ATP bound at the catalytic site of E2, whose affinity for ATP is about 30-fold less than that of E1, accelerates this conformational transition.

Adenosine Triphosphatases↗

PubMed1978

Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site.

The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.

4-Chloro-7-nitrobenzofurazan↗

PubMed2001

Photochemical generation of nitric oxide from 6-nitrobenzo[a]pyrene.

Photolabile 6-nitrobenzo[a]pyrene (6-nitroBaP) released nitric oxide (NO) under visible-light irradiation. The generation of NO and the concomitant formation of the 6-oxyBaP radical were confirmed by ESR. BaP quinones were also detected as further oxidized products of the 6-oxyBaP radical. No such photodegradation was observed with other nitrated BaPs, such as 1-nitroBaP and 3-nitroBaP. DNA-strand breakage, caused by photoexcited 6-nitroBaP, was closely related to its NO-releasing activity. MO calculations of nitrated BaP suggest that the perpendicular conformation of the nitro substituent to the aromatic ring is important for the release of NO with light. These findings may be useful for the development of a new type of NO donor.

Benzopyrenes↗

PubMed1999

Nucleophilic Substitution Reactions of Thiophenyl 4-Nitrobenzoates with Pyridines in Acetonitrile.

The kinetics of reactions between Z-thiophenyl 4-nitrobenzoates and X-pyridines in acetonitrile at 55.0 degrees C are investigated. The Brönsted plots obtained for the pyridinolysis of thiophenyl benzoates are curved, with the center of curvature at pK(a) approximately 4.2 (pK(a)(0)). The Brönsted plots for these nucleophilic reactions show a change in slope from a large (beta(X) congruent with 0.64-0.72) to a small (beta(X) congruent with 0.19-0.23) value, which can be attributed to a change in the rate-determining step from breakdown to formation of a tetrahedral intermediate in the reaction path as the basicity of the pyridine nucleophile increases. This mechanism is supported by the change of the cross-interaction constant rho(XZ) from a large positive (rho(XZ) = 1.41) for the weakly basic pyridines to a small negative (rho(XZ) = -0.32) value for the strongly basic pyridines. The reactivity-selectivity principle (RSP) holds for the rate-limiting breakdown but fails for the formation of the intermediate. The aminolysis of thiophenyl benzoate with deuterated 4-chlorobenzylamine catalyzed by glymes has primary deuterium kinetic isotope effects (PKIEs), k(H(cat))/k(D(cat)) congruent with 1.28-1.62.

Journal Article↗

PubMed2000

Formation of 4-methylphenanthrenes in palladium-catalyzed annulation of diethyl 2,2'-diiodo-4,4'-biphenyldicarboxylate with internal alkynes, using methyl nitrobenzoates as the methylating agent

The reaction of diethyl 2,2'-diiodo-4,4'-biphenyldicarboxylate (7) with diarylacetylenes in the presence of 3, 5-(NO(2))(2)C(6)H(3)CO(2)Me (MeDNB) or 4-(NO(2))C(6)H(4)CO(2)Me (MePNB), Pd(OAc)(2) (10 mol %), K(2)CO(3), and Bu(4)NBr, in DMF at 100 degrees C, gives 4-methyl-9,10-diaryl-2, 7-phenanthrenedicarboxylic acid diethyl esters in good yields. The methyl group at position 4 originates from the electron-deficient methyl nitrobenzoates. High regioselectivity for the annulation of 7 with nonsymmetrical diarylalkynes was observed with the selectivity controlled mainly by electronic factors rather than by steric factors. A competitive kinetic isotope study was carried out using a 1:1 mixture of MePNB and MePNB-d(3) in the annulation reaction of 7 which gave 4-methyl- and 4-(methyl-d(3))phenanthrene in a 67:33 ratio, leading to an apparent value of k(H)/k(D) = 1.26. Possible mechanisms for the methyl transferring process are discussed.

Journal Article↗

PubMed2001

An evaluation of the mutagenicity, metabolism, and DNA adduct formation of 5-nitrobenzo[b]naphtho[2,1-d]thiophene.

Thioarenes, sulfur-containing polycyclic aromatic compounds, are environmental contaminants suspected of posing human health risks. In this study, 5-nitrobenzo[b]naphtho[2,1-d]thiophene (5-nitro-BNT), a nitrated-thioarene, was examined for its mutagenicity, metabolism and subsequent formation of DNA adducts. 5-Nitro-BNT was weakly mutagenic in Salmonella typhimurium strains TA98 and TA100 without Aroclor-1254-induced rat liver S9 (S9), and its activity was increased in the presence of S9. Anaerobic metabolism of 5-nitro-BNT by S9 or xanthine oxidase (XO) produced one major metabolite, identified as 5-amino-BNT by NMR, MS, and UV spectroscopy and by comparison with an authentic standard. Aerobic S9 metabolism of 5-nitro-BNT produced a major metabolite, identified as trans-9,10-dihydroxy-9,10-dihydro-5-nitro-BNT (5-nitro-BNT-9,10-diol). Also present was a minor amount of 5-amino-BNT and trans-9,10-dihydroxy-9,10-dihydro-5-amino-BNT (5-amino-BNT-9,10-diol). DNA adduct analyses were performed using the (32)P-postlabeling assay and reversed-phase HPLC. Three major XO-derived calf thymus DNA adducts were detected. On the basis of their chromatographic mobilities, two adducts were identified as reaction products of 5-nitro-BNT with 2'-deoxyguanosine and one adduct with 2'-deoxyadenosine. Incorporation of allopurinol (a specific XO inhibitor) in the incubation mixture resulted in loss of all three adducts, confirming enzymatic mediation by XO. Aerobic S9 activation of 5-nitro-BNT with calf thymus DNA produced three adducts. On the basis of their chromatographic mobilities, two were identified as reaction products of 5-nitro-BNT with 2'-deoxyguanosine and one with 2'-deoxyadenosine. Incorporation of 1-aminobenzotriazole (a P450 inhibitor) in the incubation mixture resulted in a loss of these adducts, confirming enzymatic mediation by P450. Aerobic S9-catalyzed metabolism of 5-nitro-BNT-9,10-diol produced the same DNA adducts as observed with 5-nitro-BNT. Aerobic S9-catalyzed metabolism of 5-amino-BNT-9,10-diol produced the same deoxyadenosine-derived DNA adducts as observed with 5-nitro-BNT and 5-nitro-BNT-9,10-diol. These results provide additional information that both ring oxidation and nitroreduction are involved in the metabolism, DNA adduct formation and mutagenicity of 5-nitro-BNT.

Animals↗

PubMed2000

Kinetic studies of the reactions of some metal reconstituted metallothioneins with the electrophilic disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

Rabbit liver Pt7MT, Zn7MT, Bi7MT were reconstituted and the kinetic studies of the reactions with electrophilic disulphide 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were investigated to explore the possible mechanism of metals release from metallothioneins. It is revealed that the Pt7MT, Zn7MT react with DTNB biphasically, yielding a four-term rate law: Rate = k1f [MT] + k2f [DTNB][MT] + k1s [MT] + k2s [DTNB][MT]. Zn7MT reacts with disulphide DTNB more rapidly and has significantly greater observed rate constants ks, kf than Pt7MT. The kinetic data for Bi7MT indicate that the reaction is monophasic and the rate law is proved to be: Rate = k1 [MT] + k2 [DTNB][MT]. The observed pseudo-first order rate constants for above MTs are insensitive to pH value. Based on the available experimental data, the different kinetic behaviors of MTs reactions with electrophilic disulphide DTNB and a possible mechanism to release the coordinated metal ions are discussed.

Animals↗