Characterisation of metabolites of the putative cancer chemopreventive agent quercetin and their effect on cyclo-oxygenase activity.

Quercetin (3,5,7,3',4'-pentahydroxyflavone) is a flavone with putative ability to prevent cancer and cardiovascular diseases. Its metabolism was evaluated in rats and human. Rats received quercetin via the intravenous (i.v.) route and metabolites were isolated from the plasma, urine and bile. Analysis was by high-performance liquid chromatography and confirmation of species identity was achieved by mass spectrometry. Quercetin and isorhamnetin, the 3'-O-methyl analogue, were found in both the plasma and urine. In addition, several polar peaks were characterised as sulphated and glucuronidated conjugates of quercetin and isorhamnetin. Extension of the metabolism studies to a cancer patient who had received quercetin as an i.v. bolus showed that (Quercetin removed) isorhamnetin and quercetin 3'-O-sulphate were major plasma metabolites. As a catechol, quercetin can potentially be converted to a quinone and subsequently conjugated with glutathione (GSH). Oxidation of quercetin with mushroom tyrosinase in the presence of GSH furnished GSH conjugates of quercetin, two mono- and one bis-substituted conjugates. However, these species were not found in biomatrices in rats treated with quercetin. As cyclo-oxygenase-2 (COX-2) expression is mechanistically linked to carcinogenesis, we examined whether quercetin and its metabolites can inhibit COX-2 in a human colorectal cancer cell line (HCA-7). Isorhamnetin and its 4'-isomer tamarixetin were potent inhibitors, reflected in a 90% decrease in prostaglandin E-2 (PGE-2) levels, a marker of COX-2 activity. Quercetin was less effective, with a 50% decline. Quercetin 3- and 7-O-sulphate had no effect on PGE-2. The results indicate that quercetin may exert its pharmacological effects, at least in part, via its metabolites.

The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1.

Resveratrol is a cancer preventative agent that is found in red wine. Piceatannol is a closely related stilbene that has antileukaemic activity and is also a tyrosine kinase inhibitor. Piceatannol differs from resveratrol by having an additional aromatic hydroxy group. The enzyme CYP1B1 is overexpressed in a wide variety of human tumours and catalyses aromatic hydroxylation reactions. We report here that the cancer preventative agent resveratrol undergoes metabolism by the cytochrome P450 enzyme CYP1B1 to give a metabolite which has been identified as the known antileukaemic agent piceatannol. The metabolite was identified by high performance liquid chromatography analysis using fluorescence detection and the identity of the metabolite was further confirmed by derivatisation followed by gas chromatography-mass spectrometry studies using authentic piceatannol for comparison. This observation provides a novel explanation for the cancer preventative properties of resveratrol. It demonstrates that a natural dietary cancer preventative agent can be converted to a compound with known anticancer activity by an enzyme that is found in human tumours. Importantly this result gives insight into the functional role of CYP1B1 and provides evidence for the concept that CYP1B1 in tumours may be functioning as a growth suppressor enzyme.

Analysis of S-phenyl-L-cysteine in globin as a marker of benzene exposure.

An assay has been developed to determine S-phenylcysteine (SPC) in globin as a potential biomarker for exposure to benzene. The sensitivity of the assay is less than 20 pmol SPC g(-1) globin. Following acidic hydrolysis of the protein, the modified amino acid is purified by reverse phase cartridge chromatography and HPLC, prior to conversion to the tert-butyldimethylsilyl derivative and GC-MS selected ion recording. Quantitation is achieved using the internal standard [(2)H5]-SPC, and calibration lines were established using a synthetic peptide Leu-His-SPC-Asp-Lys. Control human globin was found to contain ca 30 pmol SPC g(-1) globin in two populations. The source of the apparent background level of SPC is unknown.

Application of the high-performance liquid chromatographic method for separation, purification and characterisation of p-bromophenylacetylurea and its metabolites.

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N'-hydroxy-p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N'-methyl-p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.

In vivo metabolism of the antitumor imidazoacridinone C1311 in the mouse and in vitro comparison with humans.

C1311 has emerged as the lead compound from a novel group of anticancer agents, the imidazoacridinones, and will be entering clinical trials shortly. Previous murine pharmacokinetic studies have shown C1311 to be rapidly and extensively distributed into tissues including tumor. This study has identified two major metabolites of C1311 and describes their pharmacokinetics in mice. M1 is a glucuronide of the parent compound with high concentrations in both plasma and liver. Calculated area under the plasma concentration versus time curve values were 6-fold and 2-fold greater, respectively, than C1311. Based on these studies, we propose M2 to be a nonfluorescent oxidation product because electrospray ionization-mass spectroscopy/mass spectroscopy analysis gave a molecular ion at m/z 367, 16 U greater than the parent compound. It formed rapidly in liver preparations in vitro, both murine and human, by a cytosolic process in the presence of NADPH and in vivo was detected in liver tissues at concentrations equivalent to those of C1311 but was not detectable in plasma. Preliminary in vitro toxicity studies showed M2 to be as potent as C1311 against MAC15A tumor cells. Over the first 24 h, 39% of the administered dose is eliminated via the bile (28%) mostly as C1311 or the kidneys (11%) as the glucuronide (M1). This study has given valuable information as to the likely metabolic pathway to occur in humans, and the cytotoxic metabolite M2 may play a role in the antitumor activity or toxicity of C1311 in the clinic.

Analysis of urinary and faecal porphyrin excretion patterns in human porphyrias by fast atom bombardment mass spectrometry.

We report a new method for obtaining urinary and faecal porphyrin excretion patterns in human porphyrias based on fast atom bombardment mass spectrometry (FAB-MS). Porphyrins were esterified and extracted from urine or faeces as their methyl esters for analysis by FAB-MS. The protonated pseudo-molecular ion [M + H]+ observed for each porphyrin is characteristic of that porphyrin, thus allowing a mixture of porphyrins to be analysed without the need for chromatographic separation. By using tandem MS, identification and characterisation of unknown porphyrins can be achieved. The urinary and faecal porphyrin excretion patterns from various porphyric patients obtained by FAB-MS are in good agreement with those analysed by TLC or HPLC methods.

Capacity of rat brain to metabolize m-dinitrobenzene: an in vitro study.

m-dinitrobenzene (m-DNB) is a neurotoxin producing selective brain lesions, but the in situ metabolic fate of m-DNB in brain is unknown. In this study, nitroreductive capacity of brain towards m-dinitrobenzene (m-DNB) has been investigated. Tissue slices from F344 rat brain stem, forebrain, and liver were separately incubated with 0.2 mM m-DNB. m-DNB and its metabolites were detected by HPLC, and identified by either HPLC or Mass Spectrometry (MS). All three types of tissues showed metabolic activity towards m-DNB. Metabolic disposal of m-DNB was 1.05 +/- 0.11 mumol/g wet weight/h in liver, 0.49 +/- 0.05 in brain stem, and 0.44 +/- 0.05 in forebrain (mean +/- SD, n = 4). m-Nitroaniline was found to be the main metabolite produced by both brain and liver slices, representing 57-66% of the disposal of m-DNB. Liver slices also produced 2(or 4)-amino-4(or 2)-nitrophenol, which was not detected in brain slices. We detected nitrosonitrobenzene in the slices from both parts of brain, but not in liver slices. The glucose consumption of brain slices from both areas were significantly increased in the presence of m-DNB: by 26% in the brain stem (p < 0.001) and by 17.9% in cerebral cortex (p < 0.01). This may be considered a pre-cytotoxic effect. The results demonstrate that brain has considerable nitroreductive capacity towards m-DNB, and that in situ reduction of m-DNB may be responsible for its neurotoxicity.

Determination of cis-thymine glycol in DNA by gas chromatography-mass spectrometry with selected ion recording and multiple reaction monitoring.

A novel method for the determination of cis-thymine glycol in DNA has been developed, using gas chromatography-mass spectrometry with selected ion recording or multiple reaction monitoring. The procedure involves acidic hydrolysis of DNA in the presence of the internal standard cis-[2H3]thymine glycol, followed by derivatisation with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. The method was validated on DNA that had been oxidatively modified in vitro by radiation treatment, and was then applied to determine cis-thymine glycol in human placental DNA. Background levels of 5.45+/-2.98 ng cis-thymine glycol/mg DNA were observed in the human samples.

On-line high-performance liquid chromatographic-electrospray ionization mass spectrometric method for the study of tamoxifen metabolism.

An on-line high-performance liquid chromatographic (HPLC)-electrospray ionization mass spectrometric (ESI-MS) method has been developed and optimized for the study of tamoxifen metabolism. Metabolism in mouse liver microsomes was chosen to demonstrate the applicability and superiority of the method, since mice metabolize tamoxifen faster and produce more metabolites than rats or humans. Mouse liver microsomal preparations were incubated with tamoxifen in the presence of NADPH and MgCl2. The metabolites formed were separated and analyzed by the optimized HPLC-ESI-MS system. The separation was performed on a Res Elute-BD column (5 microns particle size, 250 x 4.6 mm I.D.) with 70% (v/v) methanol in 0.5 M ammonium acetate as the mobile phase. A total of eleven metabolites have been detected, some of which have not been previously reported. The metabolites identified are: tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen, 4'-hydroxytamoxifen, 4-hydroxytamoxifen N-oxide, 4'-hydroxytamoxifen N-oxide, 4-hydroxy-N-desmethylamoxifen, 4'-hydroxy-N-desmethyltamoxifen, 3,4-dihydroxytamoxifen, 3,4-epoxytamoxifen and 3,4-epoxytamoxifen N-oxide.

Characterization of 2-amino-1-benzylbenzimidazole and its metabolites using tandem mass spectrometry.

We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocycle, 2-amino-1-benzylbenzimidazole (ABB). Three major metabolites were isolated and structurally characterized, using a combination of off-line HPLC, in conjunction with both electron ionization and fast atom bombardment ionization tandem mass spectrometry. ABB was shown to be debenzylated to afford 2-aminobenzimidazole (AB), as well as N- and C-oxidized to give 1-benzyl-N2-hydroxyaminobenzimidazole (BHB) and 2-amino-1-benzyl-hydroxybenzimidazole, respectively. The possible reasons for formation of the exocyclic hydroxylamine BHB are discussed. Furthermore, ABB is proposed as a suitable model compound for investigating parameters that control formation of toxic hydroxylamines derived from amino-azaheterocycles.

Urinary porphyrin profiles by laser desorption/ionization time-of-flight mass spectrometry without the use of classical matrices.

A method is described for the rapid analysis of mixtures of porphyrin methyl esters using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS). Ions corresponding to [M+H]+ for each component have been observed without the need for added matrix. LDI-TOF MS has been applied to the analysis and characterization of porphyrin methyl esters derived from urine samples of patients suffering from various porphyrias. This technique has been shown to provide "fingerprint" profiles of the porphyrins present in each urine sample, characteristic of the porphyria, which compare favourably with those obtained with the established method of high-performance liquid chromatography. LDI-TOF MS is a rapid and reliable method for the screening of urine from patients suffering from porphyria.

Characterization of N-benzylcarbazole and its metabolites from microsomal mixtures by tandem mass spectrometry.

The metabolism of N-benzylcarbazole (NBC) was studied in vitro using hamster hepatic microsomes to establish whether the corresponding amide is formed. This work was carried out in order to see if the extremely low pka characteristic of such a benzylic amine would allow the formation of the carbonyl derivative. No amide formation was observed. However, a number of metabolic products were detected using HPLC, including the oxidative debenzylation products, namely carbazole and benzaldehyde, together with 2 phenolic isomers of NBC. These products were tentatively characterized by their UV spectra using a rapiscan UV detector connected to HPLC equipment. The structural characterization of these 4 metabolites, together with unchanged substrate, was carried out using desorption electron impact tandem mass spectrometry (DEI-MS/MS) on a hybrid instrument with EBQ1Q2 configuration.

A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes.

The metabolisms of tamoxifen in female rat, mouse and human liver microsomal preparations were compared. Rat, mouse and human liver microsomes were incubated with tamoxifen in the presence of NADPH and MgCl2 and the metabolites formed were analysed by on-line HPLC-electrospray ionization MS. The major metabolites formed by rat liver microsomes were 4-hydroxytamoxifen, 4'-hydroxytamoxifen, N-desmethyltamoxifen and tamoxifen N-oxide. In addition, two epoxide metabolites, 3,4-epoxytamoxifen and 3',4'-epoxytamoxifen, and their hydrolysed derivatives, 3,4-dihydrodihydroxytamoxifen and 3',4'-dihydrodihydroxytamoxifen, have been identified. The pattern of the main metabolites obtained with human liver microsomes resembles qualitatively that of rat liver microsomes. The major differences between rat and human liver microsomes were that the amount of hydroxylated metabolites were much lower in human and only traces of 3,4-epoxytamoxifen and the corresponding dihydrodihydroxy derivative were detected. No 3',4'-epoxytamoxifen was detected in human liver microsomes. The four major metabolites were also formed in much larger amounts and with faster rates of formation by mouse liver microsomes, though tamoxifen N-oxide clearly predominated in this species. Polar metabolites, 3,4-dihydroxytamoxifen and 4-hydroxytamoxifen N-oxide, which were undetectable in rat and human, were formed in significant amounts in mouse microsomes. As in human microsomes, there was only one epoxide metabolite, 3,4-epoxytamoxifen, produced by mouse liver microsomes at levels lower than that found in rat. The faster rate of metabolism and the production of polar metabolites may indicate the ability of mouse to detoxify tamoxifen by rapid elimination compared with rat and human. The production of a larger amount of potentially reactive epoxide metabolites in rat may be responsible for the liver carcinogenesis in this species.

An evaluation of tandem mass spectrometry in drug metabolism studies.

The use of precursor ion and constant neutral loss scanning as a means of rapidly detecting drug metabolites is evaluated. Four clinically useful drugs, namely (i) cyclophosphamide, (ii) mifentidine, (iii) cimetropium bromide and (iv) haloperidol, were subjected to microsomal incubations to afford phase I metabolites. Aside from a minor clean-up procedure involving zinc sulfate precipitation of microsomal proteins and solid-phase extraction of metabolites using a Sep-pak C-18 cartridge, the mixtures were analysed directly by fast atom bombardment tandem mass spectrometry. It is demonstrated that such screening strategies are important in detecting novel metabolites. However, there are some problems associated with only using such methods, including (i) the possibility of not detecting metabolites that undergo unusual collision-induced dissociation fragmentation pathways, (ii) the non-detection of metabolites that have undergone metabolic change at unusual sites of reactivity, and (iii) production of artifacts derived from the parent drug by the primary ionization process. Examples are discussed that highlight both the strengths and weaknesses of such an approach.

A novel aldehyde reductase with activity towards a metabolite of aflatoxin B1 is expressed in rat liver during carcinogenesis and following the administration of an anti-oxidant.

In contrast with fractions from control animals, an aldehyde reductase, which catalyses the reduction of aflatoxin B1-dihydrodiol, in the dialdehyde form at physiological pH values, to aflatoxin B1-dialcohol, is expressed in cytosolic fractions prepared from rat livers bearing pre-neoplastic lesions, or following treatment with the anti-oxidant ethoxyquin. This expression parallels the development of resistance to the toxin. Unlike the aflatoxin B1-dihydrodiol, the dialcohol does not undergo binding to protein. This enzyme activity could play a mechanistic role in hepatocarcinogenesis and chemoprotection in the rat. Correlated n.m.r. and m.s. spectra are provided in Supplementary Publication SUP 50171 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

Identification of endogenous electrophiles by means of mass spectrometric determination of protein and DNA adducts.

Monitoring exposure to alkylating agents may be achieved by quantitatively determining the adduct levels formed with nucleic acids and/or proteins. One of the most significant results arising from the application of this approach has been the discovery in control populations of "background" levels of alkylated nucleic acid bases or alkylated proteins, in particular hemoglobin (Hb). In the case of Hb, a wide variety of such adducts have been detected and quantitated by mass spectrometric techniques, with methylated, 2-carboxyethylated, and 2-hydroxyethylated modifications being most abundant. Although the source of these alkylation products is unknown, both endogenous and exogenous sources may be proposed. We have recently confirmed the presence of the N-terminal hydroxyethylvaline adduct in control human Hb using tandem mass spectrometry (MS-MS) and have now established background levels using GC-MS in more than 70 samples. Smoking raises the levels of the adduct up to 10-fold and occupational exposure to ethylene oxide up to 300-fold. Background levels of alkylated nucleic acids may be studied by analysis of N7-alkylated guanine or N3-alkylated adenine, which are excised from nucleic acids after their formation and are excreted in urine. Although the presence of some of these urinary constituents may be accounted for by their natural occurrence in RNA or diet, the endogenous or exogenous source of others is unknown. Quantitative methods using MS-MS have now been developed for five of the observed urinary alkylguanines [N7-methyl-, N2-methyl-, N2-dimethyl-, N7-(2-hydroxyethyl)-, and N2-ethylguanine].(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of metabolites of 4,4'-diaminodiphenylmethane (methylene dianiline) using liquid chromatographic and mass spectrometric techniques.

The in vitro metabolism of 4,4'-diaminodiphenylmethane (methylene dianiline, MDA) was investigated using rabbit liver microsomes. Minimal clean-up of the microsomal incubations was carried out using zinc sulphate precipitation followed by solid-phase extraction on Sep-Pak C18 cartridges. Three metabolites were detected in hepatic microsomal incubations, namely the azodiphenylmethane (azo) azoxydiphenylmethane (azoxy) and 4-nitroso-4'-aminodiphenylmethane (nitroso) compounds. The azo and azoxy metabolites were produced enzymatically whereas the nitroso compound may have been formed via a non-enzymatic process. Reversed-phase high-performance liquid chromatography-plasma spray mass spectrometry was used to initially detect these metabolites. Fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry were utilized to further structurally characterise these compounds. Comparison of mass spectral data obtained from synthesised standards with data obtained on the putative metabolites substantiated the characterisation of these compounds.

Bioanalytical applications of tandem mass spectrometry in the in vitro metabolism of the anticholinergic drug cimetropium bromide to detect differences in species metabolism.

1. In vitro metabolism of the anticholinergic drug, cimetropium bromide, was investigated using four different animal hepatic microsomal incubates derived from rat, hamster, guinea pig, and mouse livers. 2. Constant neutral loss (CNL) tandem mass spectrometry was used to detect the presence of the N-methylenecyclopropyl-scopine functionality by monitoring loss of 54 daltons (corresponding to loss of methylenecyclopropane) in microsomal incubates. 3. A CNL loss of 46 daltons was used to screen for the presence of ester hydrolysis products. 4. A comparison of the daughter ion spectra obtained on ions detected by CNL scanning, with daughter ion spectra of synthetic standards, determined the presence of ten metabolites of cimetropium bromide. 5. Hydroxylation of the aromatic ring in the ester side-chain was found to be the major metabolic pathway, and ester bond hydrolysis was a minor metabolic pathway. 6. N-Demethylation of the bridgehead nitrogen was observed only in rat and hamster incubates. 7. Using the method of CNL scanning it was possible to screen different animal microsomal incubates without resorting to any major purification procedures such as h.p.l.c. 8. This scanning method revealed differences between species in the metabolic pathways of cimetropium bromide.

Identification of cyclophosphamide-DNA adducts in rat embryos exposed in vitro to 4-hydroperoxycyclophosphamide.

Cyclophosphamide and other bifunctional alkylating agents are potent animal teratogens inducing a variety of malformations. Although cyclophosphamide-induced DNA damage is implicated as a primary mechanism underlying the teratogenesis initiated by cyclophosphamide, additional insights into the complex nature of the teratogenic process have been hampered by the inability to analyze the primary teratogenic lesions, i.e., cyclophosphamide-DNA adducts. Using tandem mass spectrometry, we show that the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (NOR-G) and bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-NOR-G) can be detected in the DNA of organogenesis-stage rat embryos after an in vitro exposure to an embryotoxic concentration of activated cyclophosphamide, i.e., 4-hydroperoxycyclophosphamide.