Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 mu m) at 35 degrees C under isocratic conditions using 5 mM KH(2)PO(4) buffer solution pH 6.0: methanol: diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL, plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5,4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether: n-hexane: methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r >= 0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r >= 0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis. (c) 2008 Elsevier B.V. All rights reserved.

Mutagenicity and Antimutagenicity of Baccharis dracunculifolia Extract in Chromosomal Aberration Assays in Chinese Hamster Ovary Cells

Baccharis dracunculifolia De Candole (Asteraceae), a native plant from the Brazilian ""cerrado"", is widely used in folk medicine as an anti-inflammatory agent and for the treatment of gastrointestinal diseases. B. dracunculifolia has been described as the most important plant source of propolis in southeastern Brazil, which is called green propolis due to its color. The aim of the present study was to evaluate the mutagenic and antimutagenic effects of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE) on Chinese hamster ovary cells. On one hand, the results showed a significant increase in the frequencies of chromosome aberrations at the highest Bd-EAE concentration tested (100 mu g/mL). On the other hand, the lowest Bd-EAE concentration tested (12.5 mu/mL) significantly reduced the chromosome damage induced by the chemotherapeutic agent doxorubicin. The present results indicate that Bd-EAE has the characteristics of a so-called Janus compound, that is, Bd-EAE is mutagenic at higher concentrations, whereas it displays a chemopreventive effect on doxorubicin-induced mutagenicity at lower concentrations. The constituents of B. dracunculifolia responsible for its mutagenic and antimutagenic effects are probably flavonoids and phenylpropanoids, since these compounds can act either as pro-oxidants or as free radical scavengers depending on their concentration.

Partial characterization of proteins from baru (Dipteryx alata Vog) seeds

BACKGROUND: Baru (Dipteryx alata Vog.) is a fruit distributed throughout the Brazilian savanna and contains a seed with a high protein content, whose properties have been rarely explored. The purpose of this study was to characterize this protein, especially by isolation and quantifying its fractions and measuring some of its molecular properties. RESULTS: Baru seeds contain 244 g kg(-1) protein on a dry weight basis. Solubility profiles showed a preponderance of globulins. This fraction dominated the seed composition, with 61.7 wt% of the total soluble proteins. Albumins and glutelins accounted for 14 and 3.3 wt%, respectively. SDS-PAGE resolution of albumin and globulin showed main bands with molecular weights of 84 kDa and 64,66 and 73 kDa, respectively. The total protein of the flour and the globulin showed values of in vitro digestibility of 85.59% and 90.54%, relative to casein. Total globulin produced only one chromatographic peak, both on Sepharose CL-6B gel filtration and on DEAE-cellulose ion-exchange columns, eluted at a concentration of 0.12 mol L(-1) NaCl. CONCLUSION: The baru seed had high protein content with large quantities of storage proteins. The chromatographic and solubility profiles indicate the predominance of a fraction with characteristics of a legumin-type protein. (C) 2011 Society of Chemical Industry

Evaluation of the genotoxic and antigenotoxic potential of Baccharis dracunculifolia extract on V79 cells by the comet assay

Baccharis dracunculifolia (Asteraceae), the main botanical source of green propolis, is a shrub of the Brazilian `cerrado`. In folk medicine it is used as an anti-inflammatory agent, mainly for the treatment of gastrointestinal diseases. The aim of the present study was to evaluate the genotoxic and antigenotoxic effects of B. dracunculifolia ethyl acetate extract (Bd-EAE) on Chinese hamster lung fibroblasts (V79 cells) by the comet assay. Methyl methanesulfonate (MMS; 200 mu M) was used as an inducer of DNA damage. Genotoxicity was evaluated using four different concentrations of Bd-EAE: 12.5, 25.0, 50.0 and 100.0 mu g ml(-1). Antigenotoxicity was assessed before, simultaneously, and after treatment with the mutagen. The results showed a significant increase in the frequency of DNA damage in cultures treated with 50.0 and 100.0 mu g ml(-1) Bd-EAE. Regarding its antigenotoxic potential, Bd-EAE reduced the frequency of DNA damage induced by MMS. However, this chemopreventive activity depended on the concentrations and treatment regimens used. The antioxidant activity of phenolic components present in Bd-EAE may contribute to reduce the alkylation damage induced by MMS. In conclusion, our findings confirmed the chemopreventive activity of Bd-EAE and showed that this effect occurs under different mechanism. Copyright (C) 2009 John Wiley & Sons, Ltd.

In vitro Fluoride Release and Tensile Bond Strength of a Polymeric Intra-Buccal Bioadhesive

The intra-buccal polymeric bioadhesive systems that can stay adhered to the oral soft tissues for drug programmed release, with the preventive and/or therapeutic purpose has been employed for large clinical situations. A system based on hydroxypropyl methyl cellulose/Carbopol 934`/magnesium stearate (HPMC/Cp/StMg) was developed having the sodium fluoride as active principle. This kind of system was evaluated according to its resistance to the removal by means of physical test of tensile strength. Swine buccal mucosa extracted immediately after animals` sacrifice was employed as substrate for the physical trials, to obtain 16 test bodies. Artificial saliva with or without mucin was used to involve the substrate/bioadhesive system sets during the trials. Artificial salivas viscosity was determined by means of Brookfield viscometer, showing the artificial saliva with mucin 10.0 cP, and the artificial saliva without mucin 7.5 cP. The tensile strength assays showed the following averages: for the group ""artificial saliva with mucin"" - 12.89 Pa, and for the group ""without mucin"" - 12.35 Pa. Statistical analysis showed no significant difference between the assays for both artificial salivas, and it was possible to conclude that the variable mucin did not interfered with the bioadhesion process for the polymeric devices. The device was able to release fluoride in a safe, efficient and constant way up to 8 hours.

Endophytic fungi found in association with Smallanthus sonchifolius (Asteraceae) as resourceful producers of cytotoxic bioactive natural products

Smallanthus sonchifolius is a traditional Andean plant which has been cultured mainly in Brazil, Japan and New Zealand due to its medicinal properties. A study of the endophytic fungi associated to the plant was carried out in order to characterize new cytotoxic agents. Thirty two fungal strains were isolated and submitted to cultivation and extraction producing 186 extracts. Of these, 12% displayed moderate to high cytotoxic activities and were considered promising anticancer compound sources. The ethyl acetate fractions of Nigrospora sphaerica and Phoma betae liquid fermentations contained the synergistic compounds 8-hydroxy-6-methoxy-3-methylisocoumarin and (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one which are potential compounds for drug discovery. Another isolated compound, pimara-7,15-dien-3-beta-ol diterpene is being characterized for the first time through a detailed spectroscopic analysis including GC/MS, homo- and hetero-nuclear correlated NMR experiments (HMQC, HMBC, COSY and NOEdiff) along with its optical rotation.

Effects of Commonly Used Solubilizing Agents on a Model Nerve-Muscle Synapse

Solubility represents a limiting factor when testing new compounds in animal experiments, since solubilizing agents generally have pharmacological effects that can interfere with the studied substance. Vehicles are commonly used for solubilizing certain substances including apolar and polar extracts obtained from medicinal plants. In this study, fifteen vehicles were investigated on mice neuromuscular preparations. A known in vitro neuroblocker myotoxin from Bothrops jararacussu venom, bothropstoxin-I, was used as a pharmacological tool for testing the medicinal potential of apolar and polar extracts (hexane, dichloromethane, ethyl acetate and methanol) obtained from Casearia sylvestris Sw. leaves, which in turn were used for testing their solubility and concomitantly to produce no change on basal response of indirectly stimulated mouse phrenic nerve-diaphragm preparations. Taken together in vitro biological system and extracts solubility, our results showed that dimethyl sulphoxide and polyethylene glycol 400 were the better vehicles, and methanol extract solubilized on PEG 400 was the only one able to act against the paralysis induced by the myotoxin. Thus, this study points out to the relevant role that vehicles exhibit for extracting the potential pharmacological activity of plants in a given test system.

Fungal tyrosine betaine, a novel secondary metabolite from conidia of entomopathogenic Metarhizium spp. fungi

Fungi, including the entomopathogenic deuteromycete Metarhizium anisopliae, produce a wide diversity of secondary metabolites that either can be secreted or stored in specific developmental structures, e.g., conidia. Some secondary metabolites, such as pigments, polyols and mycosporines, are associated with pathogenicity and/or fungal tolerance to several stress-inducing environmental factors, including temperature and solar radiation extremes. Extracts of M. anisopliae var. anisopliae (strain ESALQ-1037) conidia were purified by chromatographic procedures and the isolated compounds analyzed by (1)H and (13)C nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. LC-MS analyses were carried out to search for mycosporines (the initial targets), but no compounds of this class were detected. A molecule whose natural occurrence was previously undescribed was identified. It consists of betaine conjugated with tyrosine, and the structure was identified as 2-([1-carboxy-2-(4-hydroxyphenyl)ethyl]amino)-N,N,N-trimethyl-2-oxoethanammonium. mannitol was the predominant compound in the alcoholic conidial extract, but no amino acids other than tyrosine were found to be conjugated with betaine in conidia. The fungal tyrosine betaine was detected also in conidial extracts of three other M. anisopliae var. anisopliae (ARSEF 1095, 5626 and 5749) and three M. anisopliae var. acridum isolates (ARSEF 324, 3391 and 7486), but it was not detected in Aspergillus nidulans conidial extract (ATCC 10074). (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

In vitro Antitumour Activity of Orsellinates

Lichen phenolic compounds exhibit antioxidant, antimicrobial, antiproliferative. and cytotoxic activities. The purpose of this study was to evaluate the anticancer activity of lecanoric acid, a secondary metabolite of the lichen Parmotrema tinctorum, and its derivatives, orsellinates, obtained by structural modification. A cytotoxicity assay was carried out hi vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma, MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity index of the compounds. n-Butyl orsellinate was the most active compound, with IC(50) Values (the concentration that inhibits 50% of growth) ranging from 7.2 to 14.0 mu g/ml, against all the cell lines tested. The compound was more active (IC(50), = 11.4 mu g/mL) against B16-F10 cells than was cisplatin (12.5 mu g/mL). Conversely, lecanoric acid and methyl orsellinate were less active against all cell lines, having an IC(50) value higher than 50 mu g/mL. Ethyl orsellinate was more active against HEp-2 than against MCF7, 786-0, or B16-F10 cells. The same pattern was observed for n-propyl and n-butyl orsellinates. n-Pentyl orsellinate was less active than n-propyl or n-butyl orsellinates against HEp-2 cells. The orsellinate activity increased with chain elongation (from methyl to n-butyl), a likely consequence of an increase in lipophilicity. The results revealed that the structural modification of lecanoric acid increases the cytotoxic activity of the derivatives tested.

Free volume and water uptake in a copolymer hydrogel series

A copolymer of X-hydroxyethyl methacrylate (HEMA) with 2-ethoxy ethyl methacrylate (EEMA) was synthesized and the molecular mobility, free volume, and density properties examined as a function of composition. These properties were correlated with the equilibrium water uptake in order to determine which of the properties were most influential in causing high water sorption, as these materials are suitable candidates for hydrogel systems. It was found that the polar HEMA repeat unit results in a rigid, glassy sample at room temperature due to the high degree of hydrogen bonding between chains whereas high EEMA content leads to rubbery samples with subambient glass transition temperatures. The free volume properties on the molecular scale measured by positron annihilation Lifetime spectroscopy (PALS) showed that higher HEMA content led to smaller, fewer holes and a lower free volume fraction than EEMA. Therefore the high water uptake of HEEMA-containing copolymers is largely related to the high polarity of the HEMA unit compared to EEMA, despite the low content of free volume into which the water can initially diffuse. Trends in density with copolymer composition, as measured on a macroscopic level, differs to that seen by PALS and indicates that the two techniques are measuring different scales of packing. (C) 1998 John Wiley & Sons, Inc.

Validity of (-)-[H-3]-CGP 12177A as a radioligand for the 'putative beta(4)-adrenoceptor' in rat atrium

1 We have recently suggested the existence in the heart of a 'putative beta(4)-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta(1)- and Bz-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[H-3]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[H-3]-CGP 12177A for this purpose for two reasons, because it is a nonconventional partial agonist and also because it is a hydrophilic radioligand. 2 Increasing concentrations of(-)-[H-3]-CGP 12177A, in the absence or presence of 20 mu M (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta(1)- and beta(2)-adrenoceptors (pK(D) 9.4+/-0.1, B-max 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta(4)-adrenoceptor' (pK(D) 7.5+/-0.1, B-max 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta(1)- and beta(2)-adrenoceptors, (-)-[H-3]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6+/-0.1, B-max 50.8+/-7.4 fmol mg(-1) protein). 3 The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3+/-0.2), (+/-)-cyanopindolol (pK(i) 7.6+/-0.2), (-)-pindolol (pK(i) 6.6+/-0.1) and (+)-carazolol (pk(i), 7.2+/-0.2) and the antagonist (-)-bupranolol (pK(i) 6.6+/-0.2), all competed for (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta(4)-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4 The catecholamines competed with (-)-[H-3]-CGP 12177A at the 'putative beta(4)-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pK(iH) 6.3 +/- 0.3, pK(i), 3.5 +/- 0.1), (-)-adrenaline (pK(iH) 6.5 +/- 0.2, pK(iL) 2.9 +/- 0.1), (-)-isoprenaline (pK(iH) 6.2 +/- 0.5, pK(iL) 3.3 +/- 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK(i) 5.5 +/- 0.1). 5 The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta(4)-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[H-3]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 +/- 0.2) or presence of GTP (pK(i) 7.3 +/- 0.2). (-)-Isoprenaline competed with (-)-[H-3]-CGP 12177A for two states in the absence (pK(iH) 6.6 +/- 0.3, pK(iL) 3.5 +/- 0.1; % H 25 +/- 7) or presence of GTP (pK(iH) 6.2 +/- 0.5, pK(iL) 3.4 +/- 0.1; % H 37 +/- 6). In contrast, at beta(1)-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6 The specificity of binding to the 'putative beta(4)-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta(4)-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxy]acetic acid, 6 mu M), SR 58611A (ethyl((7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl-2-yloxy) acetate hydrochloride, 6 mu M), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)ethan-1-ol, 60 mu M), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 mu M) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 mu M) caused less than 22% inhibition of (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 mu M), phentolamine (10 mu M), 5-HT(100 mu M) and the 5-HT4 receptor antagonist SE 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1 ,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7 Non-conventional partial agonists, the antagonist (-)-bupranolol and catecholamines all competed for (-)-[H-3]-CGP 12177A binding in the absence of (-)-propranolol at beta(1)-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative beta(4)-adrenoceptor'. 8 We have established and validated a radioligand binding assay in rat atrium for the 'putative beta(4)-adrenoceptor' which is distinct from beta(1)-, beta(2)- and beta(3)-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta(4)-adrenoceptor'.

Sigmosceptrins A-C: New norditerpenes from a southern Australian marine sponge, Sigmosceptrella sp.

A Sigmosceptrella sp. of sponge collected during trawling operations in the Great Australian Eight, Australia, has yielded a series of new norterpenes. These include a new bisnorditerpene, sigmosceptrin-A (5); two new norditerpenes, sigmosceptrin-B (14) and sigmosceptrin-C (15), isolated as their methyl esters (6) and (7) respectively; and an ethylated artefact, sigmosceptrin-B ethyl ester (8). Complete stereostructures were assigned to the sigmosceptrins by spectroscopic analysis, chemical degradation, derivatization, and by a single-crystal X-ray structural analysis. A biosynthetic pathway is proposed that requires a common biosynthetic precursor to both the sigmosceptrins and norterpene cyclic peroxides.

Hydromorphone-3-glucuronide: Biochemical synthesis and preliminary pharmacological evaluation

Hydromorphone-3-glucuronide (H3G) was synthesized biochemically using rat liver microsomes, uridine-5'-diphosphoglucuronic acid (UDPGA) and the substrate, hydromorphone. Initially, the crude putative H3G product was purified by ethyl acetate precipitation and washing with acetonitrile, Final purification was achieved using semi-preparative high-performance-liquid-chromatography (HPLC) with ultraviolet (UV) detection. The purity of the final H3G product was shown by HPLC with electrochemical and ultraviolet detection to be > 99.9% and it was produced in a yield of approximate to 60% (on a molar basis). The chemical structure of the putative H3G was confirmed by enzymatic hydrolysis of the glucuronide moiety using P-glucuronidase, producing a hydrolysis product with the same HPLC retention time as the hydromorphone reference standard. Using HPLC with tandem mass spectrometry (HPLC-MS-MS) in the positive ionization mode, the molecular mass (M+1) was found to be 462 g/mol, in agreement with H3G's expected molecular weight of 461 g/mol. Importantly, proton-NMR indicated that the glucuronide moiety was attached at the 3-phenolic position of hydromorphone. A preliminary evaluation of H3G's intrinsic pharmacological effects revealed that following icy administration to adult male Sprague-Dawley rats in a dose of 5 mu g, H3G evoked a range of excitatory behavioural effects.including chewing, rearing, myoclonus, ataxia and tonic-clonic convulsions, in a manner similar to that reported previously for the glucuronide metabolites of morphine, morphine-3-glucuronide and normorphine-3-glucuronide.

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography

A simple method for the measurement of pindolol enantiomers by HPLC is presented. Alkalinized serum or urine is extracted with ethyl acetate and the residue remaining after evaporation of the organic layer is then derivatised with (S)-(-)-alpha-methylbenzyl isocyanate. The diastereoisomers of derivatised pindolol and metoprolol (internal standard) are separated by high-performance liquid chromatography (HPLC) using a C-18 silica column and detected using fluorescence (excitation lambda: 215 nm, emission lambda: 320 nm). The assay displays reproducible linearity for pindolol enantiomers with a correlation coefficient of r(2) greater than or equal to 0.998 over the concentration range 8-100 ng ml(-1) for plasma and 0.1-2.5 mu g ml(-1) for urine. The coefficient of variation for accuracy and precision of the quality control samples for both plasma and urine are consistently

Substrate-dependent regulation of human arylamine N-acetyltransferase-1 in cultured cells

Arylamine N-acetyltransferase-1 (NAT1) is a polymorphically expressed enzyme that is widely distributed throughout the body. In the present study, we provide evidence for substrate-dependent regulation of this enzyme. Human peripheral blood mononuclear cells cultured in medium supplemented with p-aminobenzoic acid (PABA; 6 mu M) for 24 h showed a significant decrease (50-80%) in NAT1 activity. The loss of activity was concentration-dependent (EC50 similar to 2 mu M) and selective because PABA had no effect on the activity of constitutively expressed lactate dehydrogenase or aspartate aminotransferase. PABA also induced down-regulation of NAT1 activity in several human cell lines grown at confluence. Substrate-dependent downregulation was not restricted to PABA. Addition of other NAT1 substrates, such as p-aminosalicylic acid, ethyl-p-aminobenzoate, or p-aminophenol to peripheral blood mononuclear cells in culture also resulted in significant (P < .05) decreases in NAT1 activity. However, addition of the NAT2-selective substrates sulfamethazine, dapsone, or procainamide did not alter NAT1 activity. Western blot analysis using a NAT1-specific antibody showed that the loss of NAT1 activity was associated with a parallel reduction in the amount of NAT1 protein (r(2) = 0.95). Arylamines that did not decrease NAT1 activity did not alter NAT1 protein levels. Semiquantitative reverse transcriptase polymerase chain reaction of mRNA isolated from treated and untreated cells revealed no effect of PABA on NAT1 mRNA levels. We conclude that NAT1 can be down-regulated by arylamines that are themselves NAT1 substrates. Because NAT1 is involved in the detoxification/activation of various drugs and carcinogens, substrate-dependent regulation may have important consequences with regard to drug toxicity and cancer risk.