Rapid and directly interpretable antimicrobial susceptibility profiling by continuous microvolume-electroanalysis of ferricyanide-mediated bacterial respiration.

We present a novel method for the electroanalysis of potassium ferricyanide-mediated bacterial electron transport, to rapidly assess viability and construct interpretable antimicrobial susceptibility profiles. Electrochemical minimum inhibitory concentrations (ecMICs) became determinable with a high correlation to the results from conventional assays.

Effect of ε-Poly-L-lysine on a Glucose Sensor Based on Glucose Oxidase and Ferricyanide Ion.

ε-Poly-L-lysine (εPL) is a homopolymer of L-lysine residues with linkages between the α-carboxyl and ε-amino groups. εPL exists as a polycationic species under acidic and neutral conditions, and can form a polyion complex with negatively charged glucose oxidase (GOx). We previously reported that εPL significantly promoted a GOx enzymatic reaction using ferricyanide ion ([Fe(CN)6]3-) as the oxidant. Here, we construct a GOx-immobilized electrode using εPL and glutaraldehyde, and show that [Fe(CN)6]3- can efficiently act as the mediator in this electrode. Ferricyanide ion failed to function adequately as the mediator when bovine serum albumin or other polyamines (e.g., polyallylamine, α-poly-L-lysine) were used instead of εPL. The GOx-immobilized electrode using εPL successfully responded to glucose even when the [Fe(CN)6]3- concentration was as low as 1 mM, and exhibited a wide dynamic range of up to several tens of mM. Thus, εPL is considered to be a useful additive for glucose sensors based on the [Fe(CN)6]4-/3--mediated GOx catalytic current.

Removal of the H subunit results in enhanced exposure of the semiquinone sites in the LM dimer from Rhodobacter sphaeroides to oxidation by ferricyanide and by O2.

Bacterial reaction centers (RC) from Rhodobacter sphaeroides have been widely used to functionalize electrodes and to generate photocurrent. However, in most studies, direct electron transfer from the semiquinone to the electrode was not observed because the H subunit of the RC shields the semiquinone. It is demonstrated in the current work that removal of the H subunit effectively exposes the semiquinone sites in the LM dimer. This is demonstrated by measuring the second-order rate constant for the reaction between ferricyanide and the anionic semiquinone Q A- formed by an actinic flash. The rate constant increases 1000-fold for Q A- oxidation by ferricyanide in the LM dimer compared to the intact RC. The second-order rate constant approaches the diffusion limit of 6 × 109 M-1·s-1 at low pH, but it decreases steadily when the pH is above 6.5. This pH dependence suggests that the protonation state of the LM dimer plays an important role in controlling the electron transfer kinetics. It is also shown that the addition of exogenous ubiquinone to replenish the QB site, which is mostly empty in the LM dimer, leads to oxidation of Q A- by O2 following an actinic flash. It is concluded that removal of the H subunit results in exposure of the semiquinone sites of the LM dimer to externally added oxidants and may provide a strategy for enhancing direct electron transfer from the RC to an electrode.

Metoprolol-pridopidine drug-drug interaction and food effect assessments of pridopidine, a new drug for treatment of Huntington's disease.

AIMS: Pridopidine is an oral drug in clinical development for treatment of patients with Huntington's disease. This study examined the interactions of pridopidine with in vitro cytochrome P450 activity and characterized the effects of pridopidine on CYP2D6 activity in healthy volunteers using metoprolol as a probe substrate. The effect of food on pridopidine exposure was assessed. METHODS: The ability of pridopidine to inhibit and/or induce in vitro activity of drug metabolizing enzymes was examined in human liver microsomes and fresh hepatocytes. CYP2D6 inhibition potency and reversibility was assessed using dextromethorphan. For the clinical assessment, 22 healthy subjects were given metoprolol 100 mg alone and concomitantly with steady-state pridopidine 45 mg twice daily. Food effect on a single 90 mg dose of pridopidine was evaluated in a crossover manner. Safety assessments and pharmacokinetic sampling occurred throughout the study. RESULTS: Pridopidine was found to be a metabolism dependent inhibitor of CYP2D6, the main enzyme catalysing its own metabolism. Flavin-containing monooxygenase heat inactivation of liver microsomes did not affect pridopidine metabolism-dependent inhibition of CYP2D6 and its inhibition of CYP2D6 was not reversible with addition of FeCN3 . Exposure to metoprolol was markedly increased when coadministered with pridopidine; the ratio of the geometric means (90% confidence interval) for maximum observed plasma concentration, and area under the plasma concentration-time curve from time 0 to the time of the last quantifiable concentration and extrapolated to infinity were 3.5 (2.9, 4.22), 6.64 (5.27, 8.38) and 6.55 (5.18, 8.28), respectively. Systemic exposure to pridopidine was unaffected by food conditions. CONCLUSIONS: As pridopidine is a metabolism-dependent inhibitor of CYP2D6, systemic levels of drugs metabolized by CYP2D6 may increase with chronic coadministration of pridopidine. Pridopidine can be administered without regard to food.

Scanning electrochemical microscopy based evaluation of influence of pH on bioelectrochemical activity of yeast cells - Saccharomyces cerevisiae.

In this research scanning electrochemical microscopy was applied for the investigation of immobilized yeast Saccharomyces cerevisiae cells. Two redox mediators based system was applied in order to increase the efficiency of charge transfer from yeast cells. 9,10-phenanthrenequinone (PQ) was applied as a lipophilic redox mediator, which has the ability to cross the cell's membrane; another redox mediator was ferricyanide, which acted as a hydrophylic electron acceptor able to transfer electrons from the PQ to the working electrode of SECM. Hill's function was applied to determine the optimal pH for this described SECM-based system. The influence of pH on cell viability could be well described by Hill's function. It was determined that at pH 6.5 the PQ has a minimal toxic influence on yeast cells, and the kinetics of metabolic processes in cells as well as electron transfer rate achieved in consecutive action of both redox mediators were appropriate to achieve optimal current signals.

Trivalent metal ions based on inorganic compounds with in vitro inhibitory activity of matrix metalloproteinase 13.

Collagenase-3 (MMP-13) inhibitors have attracted considerable attention in recent years and have been developed as a therapeutic target for a variety of diseases, including cancer. Matrix metalloproteinases (MMPs) can be inhibited by a multitude of compounds, including hydroxamic acids. Studies have shown that materials and compounds containing trivalent metal ions, particularly potassium hexacyanoferrate (III) (K3[Fe(CN)6]), exhibit cdMMP-13 inhibitory potential with a half maximal inhibitory concentration (IC50) of 1.3µM. The target protein was obtained by refolding the recombinant histidine-tagged cdMMP-13 using size exclusion chromatography (SEC). The secondary structures of the refolded cdMMP-13 with or without metal ions were further analyzed via circular dichroism and the results indicate that upon binding with metal ions, an altered structure with increased domain stability was obtained. Furthermore, isothermal titration calorimetry (ITC) experiments demonstrated that K3[Fe(CN)6]is able to bind to MMP-13 and endothelial cell tube formation tests provide further evidence for this interaction to exhibit anti-angiogenesis potential. To the best of our knowledge, no previous report of an inorganic compound featuring a MMP-13 inhibitory activity has ever been reported in the literature. Our results demonstrate that K3[Fe(CN)6] is useful as a new effective and specific inhibitor for cdMMP-13 which may be of great potential for future drug screening applications.

Direct Recording of Trans-Plasma Membrane Electron Currents Mediated by a Member of the Cytochrome b561 Family of Soybean.

Trans-plasma membrane electron transfer is achieved by b-type cytochromes of different families, and plays a fundamental role in diverse cellular processes involving two interacting redox couples that are physically separated by a phospholipid bilayer, such as iron uptake and redox signaling. Despite their importance, no direct recordings of trans-plasma membrane electron currents have been described in plants. In this work, we provide robust electrophysiological evidence of trans-plasma membrane electron flow mediated by a soybean (Glycine max) cytochrome b561 associated with a dopamine ß-monooxygenase redox domain (CYBDOM), which localizes to the plasma membrane in transgenic Arabidopsis (Arabidopsis thaliana) plants and CYBDOM complementary RNA-injected Xenopus laevis oocytes. In oocytes, two-electrode voltage clamp experiments showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a concentration- and type-specific manner. Current amplitudes were voltage dependent, strongly potentiated in oocytes preinjected with ascorbate (the canonical electron donor for cytochrome b561), and abolished by mutating a highly conserved His residue (H292L) predicted to coordinate the cytoplasmic heme b group. We believe that this unique approach opens new perspectives in plant transmembrane electron transport and beyond.

Adherence to standard precautions from the standpoint of the Health Belief Model: the practice of recapping needles / Adesão às precauções padrão sob o prisma do Modelo de Crenças em Saúde: a prática de reencapar agulhas

The aim of this study was to applythe Health Belief Model to explain the adherence to the recommendation not to recap needles by dentists and dental assistants of the public health system in a municipality in the State of São Paulo. A questionnaire validated and adapted for the oral health area was used, which included variables related to the frequency of recapping and health beliefs using Likert-type scales. The relationship between beliefs and adherence to the recommendation not to recap needles was obtained by regression analysis. Of all the professionals in this study (n=79), the majority (83.5%) reported recapping needles at least once in the last month. Through regression analysis, it was observed that the relationship between the beliefs described by the model and the attitude whether or not to follow the recommendation not to recap needles was explained by a lower perception of psychological barriers and a greater perception of stimuli not to recap needles. The conclusion reached is that the acceptance of recommendations to prevent working accidents with biological material was explained by some dimensions of the Health Belief Model, enabling discussion about reformulation of training offered to professionals of the public health system.

Objetivou-se neste estudo aplicar o Modelo de Crenças em Saúde a fim de explicar a adesão à recomendação de não reencapar agulhas por cirurgiões-dentistas e auxiliares de saúde bucal da rede pública de um município paulista. Utilizou-se um questionário validado e adaptado para a área de saúde bucal, que contemplava variáveis relativas à frequência do reencape e crenças em saúde, por meio de escalas tipo Likert. A relação entre as crenças e a adesão à recomendação de não reencapar agulhas foi obtida por meio da análise de regressão. Da amostra de profissionais obtida por adesão ao estudo (n = 79), a maioria (83,5%) relatou ter reencapado agulhas pelo menos alguma vez no último mês. Por meio da análise de regressão, foi observado que a relação entre as crenças descritas pelo modelo e a atitude de aderir ou não à recomendação de não reencapar agulhas foi explicada por uma menor percepção de barreiras psicológicas e por uma maior percepção de estímulos para não reencapar agulhas. Conclui-se que a aceitação das recomendações para prevenir acidentes do trabalho com material biológico foi explicado por algumas dimensões do Modelo de Crenças em Saúde, possibilitando a discussão sobre a reformulação de capacitações oferecidas para profissionais do sistema público de saúde.

New synthetic method of 8-oxocoptisine starting from natural quaternary coptisine as anti-ulcerative colitis agent.

Quaternary coptisine (1), a natural bioactive quaternary protoberberine alkaloid (QPA), was treated with potassium ferricyanide in aqueous solution of 5 N sodium hydroxide leading to the acquisition of 8-oxocoptisine (2) with much higher yield than reported in the literature. This is the first report of the oxidation of a natural QPA by applying potassium ferricyanide as an oxidant. 8-Oxocoptisine showed significant anti-ulcerative colitis efficacy in vitro with EC50 value being 8.12 × 10(- 8) M.

Involvement of neuronal nitric oxide synthase in desensitisation of µ-opioid receptors in the rat locus coeruleus.

Nitric oxide (NO) has been recently shown to enhance µ-opioid receptor (MOR) desensitisation in locus coeruleus (LC) neurons. The aim of this study was to evaluate by single-unit extracellular recordings in rat brain slices whether the neuronal NO synthase is involved in MOR desensitisation in LC neurons. As expected, a high concentration of the opioid agonist Met(5)-enkephalin (ME; 10 µM, 10 min) strongly desensitised the inhibition induced by a test application of ME (0.8 µM, 1 min), whereas lower ME concentrations (1 and 3 µM) only weakly desensitised it. The neuronal NO synthase inhibitors 7-nitroindazole (10-100 µM), S-methyl-L-thiocitrulline (0.01-10 µM) and N(ω)-propyl-L-arginine (1-10 µM) attenuated ME (10 µM)-induced opioid desensitisation, although the endothelial NO synthase inhibitor N(5)-(1-iminoethyl)-L-ornithine (3-30 µM) failed to change it. The NO donor sodium nitroprusside (1 mM), but not its inactive analog potassium ferricyanide (1 mM), enhanced the ME (3 µM)-induced desensitisation and prevented the effect of S-methyl-L-thiocitrulline (10 µM). Sodium nitroprusside (1 mM) failed to change the desensitisation of α2-adrenoceptors by noradrenaline (100 µM, 10 min). These results suggest the contribution of NO and a neuronal type of NO synthase in homologous MOR desensitisation in rat LC neurons.

Endogenous nitric oxide but not exogenous no-donor S-nitroprussiate facilitates NMDA excitation in spontaneous rhythmic neonatal rat brainstem slice.

Nitric oxide (NO) is an excitatory agent within the isolated respiratory network of immature rats (Pierrefiche et al., 2002) and modulates bursting discharge of rhythmic respiratory neurone in juvenile rats (Pierrefiche et al., 2007). However, whether NO is acting directly via a specific cellular mechanism or by increasing NMDA receptor activity is unknown. Our present aim was to study NO modulation of NMDA-induced excitation within the isolated neonatal respiratory network. The NO-scavenger, haemoglobin, and the NOS inhibitor L-NO-Arg, reduced spontaneous activity and were more effective during NMDA-induced excitation. Both diethylamine-NO (DEA-NO) and S-nitroprussiate (SNP), two NO-donors not related chemically, increased spontaneous activity in a dose-dependent manner. However, when co-applied with NMDA only DEA-NO facilitated NMDA-induced excitation whereas SNP partially reversed or prevented NMDA-induced excitation. Similar reversion of NMDA-induced excitation were obtained with K3-(FeCN)6 (Fe III) or inactivated SNP. On the contrary, FeSO4 did not have any effect on either spontaneous activity or NMDA-induced excitation. These data suggest that activation of NMDA receptors increase endogenous NO production which participates in endogenous NMDA-induced excitation during spontaneous XII bursting activity. It also demonstrated that the type of NO-donors used during pharmacological study implicating NMDA receptors should be carefully chosen.

Probing the picosecond kinetics of the photosystem II core complex in vivo.

Photosystems I (PSI) and II (PSII) are two major pigment-protein complexes of photosynthetic organisms that function in series to convert sunlight energy into chemical energy. We have studied the picosecond fluorescence behaviour of the core of both photosystems in vivo by using two Synechocystis PCC 6803 mutants: BE cells contain PSI but are lacking both PSII and the light-harvesting complexes called phycobilisomes (PBs) whereas PAL cells contain both PSI and PSII but lack the PBs. Measurements were performed at room temperature and at 77 K. The fluorescence kinetics of PSI and PSII can nicely be separated, en passant providing the PSI/PSII ratio. At room temperature, the PSI kinetics are identical to those of isolated PSI whereas the PSII kinetics can equally well be described by the in vitro trap-limited model of Y. Miloslavina, M. Szczepaniak, M. G. Muller, J. Sander, M. Nowaczyk, M. Rogner and A. R. Holzwarth, Biophys J., 2009, 96(2), 621-631, and by the transfer-to-the-trap-limited model of C. D. van der Weij-de Wit, J. P. Dekker, R. van Grondelle and I. H. M. van Stokkum, J. Phys. Chem. A, 2011, 115(16), 3947-3956, albeit that the in vivo kinetics turn out to be somewhat slower. At 77 K several low-energy pigments are observed in both photosystems which complicate the overall dynamics but the PSII kinetics can still be described by both a trap-limited and a transfer-to-the-trap-limited model.

Two-dimensional fluorescence difference gel electrophoresis analysis of Listeria monocytogenes submitted to a redox shock.

The influence of redox alteration on the growth and proteomic pattern of Listeria monocytogenes was investigated. A redox shock was induced in cultures by addition of 3mM ferricyanide (FeCN) and 6mM dithiothreitol (DTT) to increase or to decrease respectively the redox potential naturally occurring at the beginning of growth. In both conditions, the reducing and oxidizing redox shock had a strong influence, decreasing the maximum growth rate by half compared to a control culture. The proteomic analysis of L. monocytogenes performed by two-dimensional difference gel electrophoresis (2D-DIGE) exhibited twenty-three proteins differentially expressed (P<0.05), among these, many were oxidoreductases, and proteins involved in cellular metabolism (glycolysis, protein synthesis), detoxification (kat) or adhesion (Lmo1634).

How do erythrocytes contribute to the ABTS* scavenging capacity of blood?

It has been suggested lately that erythrocytes contribute significantly to the oxidant scavenging capacity (OSC) of blood and that surface adsorption of polyphenols enhances the antioxidant capacity of erythrocytes. The aim of this study was to examine the contribution of erythrocytes to the OSC of whole blood measured with a substrate not penetrating into the cells. Comparison of reduction of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical (ABTS*) by whole blood and blood plasma indicates that erythrocytes do contribute to ABTS* reduction but their contribution is lower with respect to plasma. ABTS* reduction by erythrocytes and its enhancement by polyphenols were inhibited by thiol reagents (N-ethylmaleimide and iodacetate). These reagents inhibited also the reduction of extracellular ferricyanide by erythrocytes and its enhancement by polyphenols. On this basis we postulate that the contribution of erythrocytes to the blood OSC estimated by ABTS* decolorization is at least partly due to the transmembrane-reducing system, which activity is routinely assayed by ferricyanide reduction.

Molecular "light switch" for G-quadruplex DNA: cycling the switch on and off.

We report a new G-quadruplex DNA "light switch", where the light switch can be cycled on and off through the successive introduction of G-quadruplex DNA and [Fe(CN)(6)](4-) ions.

Nitrogen dioxide oxidizes mitochondrial cytochrome c.

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9-19). However, the subsequent re-evaluation of the redox potential of the NO/NO(-) couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO(2)(•) a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3×10(6)M(-1)s(-1) for the reaction of NO(2)(•) with ferrocytochrome c. The importance of NO(2)(•) was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO(2)(•) into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO(-) or HNO) or of peroxynitrite.

On the role of ß-cyanoalanine synthase (CAS) in metabolism of free cyanide and ferri-cyanide by rice seedlings.

A study was conducted to investigate the contribution of ß-cyanoalanine synthase (CAS) to the botanical metabolism of free cyanide and iron cyanides. Seedlings of rice (Oryza sativa L. cv. XZX 45) were grown hydroponically and then amended with free cyanide (KCN) or ferri-cyanide [K(3)Fe(CN)(6)] into the growth media. Total cyanide, free cyanide, and Fe(3+)/Fe(2+) in aqueous solution were analyzed to identify the speciation of K(3)Fe(CN)(6). Activity of CAS in different parts of the rice seedlings was also assayed in vivo and results indicated that dissociation of K(3)Fe(CN)(6) to free cyanide in solution was negligible. Almost all of the applied KCN was removed by rice seedlings and the metabolic rates were concentration dependent. Phyto-transport of K(3)Fe(CN)(6) was apparent, but appreciable amounts of cyanide were recovered in plant tissues. The metabolic rates of K(3)Fe(CN)(6) were also positively correlated to the concentrations supplied. Rice seedlings exposed to KCN showed a considerable increase in the CAS activity and roots had higher CAS activity than shoots, indicating that CAS plays an important role in the botanical assimilation of KCN. However, no measurable change of CAS activity in different parts of rice seedlings exposed to K(3)Fe(CN)(6) was detected, suggesting that K(3)Fe(CN)(6) is likely metabolized by rice directly through an unknown pathway rather than the ß-cyanoalanine pathway.

Sodium nitroprusside may modulate Escherichia coli antioxidant enzyme expression by interacting with the ferric uptake regulator.

Efforts to explore possible relationships between nitric oxide (NO) and antioxidant enzymes in an Escherichia coli model have uncovered a possible interaction between sodium nitroprusside (SNP), a potent, NO-donating drug, and the ferric uptake regulator (Fur), an iron(II)--dependent regulator of antioxidant and iron acquisition proteins present in Gram-negative bacteria. The enzymatic profiles of superoxide dismutase and hydroperoxidase during logarithmic phase of growth were studied via non-denaturing polyacrylamide gel electrophoresis and activity staining specific to each enzyme. Though NO is known to induce transcription of the manganese-bearing isozyme of SOD (MnSOD), treatment with SNP paradoxically suppressed MnSOD expression and greatly enhanced the activity of the iron-containing equivalent (FeSOD). Fur, one of six global regulators of MnSOD transcription, is uniquely capable of suppressing MnSOD while enhancing FeSOD expression through distinct mechanisms. We thus hypothesize that Fur is complacent in causing this behaviour and that the iron(II) component of SNP is activating Fur. E. coli was also treated with the SNP structural analogues, potassium ferricyanide (PFi) and potassium ferrocyanide (PFo). Remarkably, the ferrous PFo was capable of mimicking the SNP-related pattern, whereas the ferric PFi was not. As Fur depends upon ferrous iron for activation, we submit this observation of redox-specificity as preliminary supporting evidence for the hypothesized Fur-SNP interaction. Iron is an essential metal that the human innate immune system sequesters to prevent its use by invading pathogens. As NO is known to inhibit iron-bound Fur, and as activated Fur regulates iron uptake through feedback inhibition, we speculate that the administration of this drug may disrupt this strategic management of iron in favour of residing Gram-negative species by providing a source of iron in an otherwise iron-scarce environment capable of encouraging its own uptake. However, these gains may be counteracted by the oxidative consequences of iron and NO, as the former can catalyse the formation of toxic free radical species while the latter can inhibit enzymes and contribute to the formation of other toxic compounds. The potential consequences of SNP on microbial growth warrant future investigation.

Factor affecting the endogenous ß-glucuronidase activity in rapeseed haploid cells: how to avoid interference with the Gus transgene in transformation studies.

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous ß-glucuronidase which interferes with the expression of bacterial ß-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24°C, 38°C and 55°C) were investigated. To avoid this problem in microspores, two incubation temperatures (38°C and 55°C) at different periods after GUS assay (4, 24 and 48h) and in the presence of 1mM potassium ferricyanide and 1mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1mM potassium ferricyanide and 1mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55°C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2µM gibberellic acid.

Metabolic intermediate complex formation of human cytochrome P450 3A4 by lapatinib.

Lapatinib, an oral breast cancer drug, has recently been reported to be a mechanism-based inactivator of cytochrome P450 (P450) 3A4 and also an idiosyncratic hepatotoxicant. It was suggested that formation of a reactive quinoneimine metabolite was involved in mechanism-based inactivation (MBI) and/or hepatotoxicity. We investigated the mechanism of MBI of P450 3A4 by lapatinib. Liquid chromatography-mass spectrometry analysis of P450 3A4 after incubation with lapatinib did not show any peak corresponding to irreversible modifications. The enzymatic activity inactivated by lapatinib was completely restored by the addition of potassium ferricyanide. These results indicate that the mechanism of MBI by lapatinib is quasi-irreversible and mediated via metabolic intermediate complex (MI complex) formation. This finding was verified by the increase in a signature Soret absorbance at approximately 455 nm. Two amine oxidation products of the metabolism of lapatinib by P450 3A4 were characterized: N-hydroxy lapatinib (M3) and the oxime form of N-dealkylated lapatinib (M2), suggesting that a nitroso or another related intermediate generated from M3 is involved in MI complex formation. In contrast, P450 3A5 was much less susceptible to MBI by lapatinib via MI complex formation than P450 3A4. In addition, P450 3A5 had a significantly lower ability than 3A4 to generate M3, consistent with N-hydroxylation as the initial step in the pathway to MI complex formation. In conclusion, our results demonstrate that the primary mechanism for MBI of P450 3A4 by lapatinib is not irreversible modification by the quinoneimine metabolite, but quasi-irreversible MI complex formation mediated via oxidation of the secondary amine group of lapatinib.