Low-dose paroxetine exposure causes lifetime declines in male mouse body weight, reproduction and competitive ability as measured by the novel organismal performance assay.

Paroxetine is a selective serotonin reuptake inhibitor (SSRI) that is currently available on the market and is suspected of causing congenital malformations in babies born to mothers who take the drug during the first trimester of pregnancy. We utilized organismal performance assays (OPAs), a novel toxicity assessment method, to assess the safety of paroxetine during pregnancy in a rodent model. OPAs utilize genetically diverse wild mice (Mus musculus) to evaluate competitive performance between experimental and control animals as they compete among each other for limited resources in semi-natural enclosures.

Regulation of CYP3A genes by glucocorticoids in human lung cells.

Inhaled glucocorticoids are the first-line treatment for patients with persistent asthma.  However, approximately thirty percent of patients exhibit glucocorticoid insensitivity, which may involve excess metabolic clearance of the glucocorticoids by CYP3A enzymes in the lung.  CYP3A4, 3A5, and 3A7 enzymes metabolize glucocorticoids, which in turn induce CYP3A genes.

Metabolism of beclomethasone dipropionate by cytochrome P450 3A enzymes.

Inhaled glucocorticoids, such as beclomethasone dipropionate (BDP), are the mainstay treatment of asthma. However, ≈ 30% of patients exhibit little to no benefit from treatment. It has been postulated that glucocorticoid resistance, or insensitivity, is attributable to individual differences in glucocorticoid receptor-mediated processes.

Fluticasone propionate pharmacogenetics: CYP3A4*22 polymorphism and pediatric asthma control.

Objective: To determine the relationship between allelic variations in genes involved in fluticasone propionate (FP) metabolism and asthma control among children with asthma managed with inhaled FP.

Study Design: The relationship between variability in asthma control scores and genetic variation in drug metabolism was assessed by genotyping 9 single nucleotide polymorphisms in the CYP3A4, CYP3A5, and CYP3A7 genes. Genotype information was compared with asthma control scores (0=well controlled to 15=poorly controlled), determined using a questionnaire modified from the National Heart Lung and Blood Institute's Expert Panel 3 guidelines.

Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes.

Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds.

Reactive intermediates produced from the metabolism of the vanilloid ring of capsaicinoids by p450 enzymes.

This study characterized electrophilic and radical products derived from the metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.

Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli.

Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E.

Conformational dynamics of CYP3A4 demonstrate the important role of Arg212 coupled with the opening of ingress, egress and solvent channels to dehydrogenation of 4-hydroxy-tamoxifen.

Background: Structure-based methods for P450 substrates are commonly used during drug development to identify sites of metabolism. However, docking studies using available X-ray structures for the major drug-metabolizing P450, CYP3A4, do not always identify binding modes supportive of the production of high-energy toxic metabolites. Minor pathways such as P450-catalyzed dehydrogenation have been experimentally shown to produce reactive products capable of forming biomolecular adducts which can lead to increased risk toxicities.

Respective roles of CYP2A5 and CYP2F2 in the bioactivation of 3-methylindole in mouse olfactory mucosa and lung: studies using Cyp2a5-null and Cyp2f2-null mouse models.

The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice.

Transient receptor potential vanilloid-1 (TRPV1) is a mediator of lung toxicity for coal fly ash particulate material.

Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1).

Quantum mechanically derived AMBER-compatible heme parameters for various states of the cytochrome P450 catalytic cycle.

Molecular mechanics (MM) methods are computationally affordable tools for screening chemical libraries of novel compounds for sites of P450 metabolism. One challenge for MM methods has been the absence of a consistent and transferable set of parameters for the heme within the P450 active site. Experimental data indicate that mammalian P450 enzymes vary greatly in the size, architecture, and plasticity of their active sites.

Contributions of TRPV1, endovanilloids, and endoplasmic reticulum stress in lung cell death in vitro and lung injury.

Endogenous agonists of transient receptor potential vanilloid-1 (TRPV1) (endovanilloids) are implicated as mediators of lung injury during inflammation. This study tested the hypothesis that endovanilloids produced following lipopolysaccharide (LPS) treatment activate TRPV1 and cause endoplasmic reticulum stress/GADD153 expression in lung cells, representing a mechanistic component of lung injury. The TRPV1 agonist nonivamide induced GADD153 expression and caused cytotoxicity in immortalized and primary human bronchial, bronchiolar/alveolar, and microvascular endothelial cells, proportional to TRPV1 mRNA expression.

Electrophilic components of diesel exhaust particles (DEP) activate transient receptor potential ankyrin-1 (TRPA1): a probable mechanism of acute pulmonary toxicity for DEP.

Inhalation of environmental particulate matter (PM) is correlated with adverse health effects in humans, but gene products that couple detection with cellular responses, and the specific properties of PM that target different pathways, have not been fully elucidated. TRPA1 and V1 are two cation channels expressed by sensory neurons and non-neuronal cells of the respiratory tract that have been implicated as possible mediators of PM toxicity. The goals of this research were to determine if environmental PM preferentially activated TRPA1 and to elucidate the criteria responsible for selectivity.

Structure-activity relationship of capsaicin analogs and transient receptor potential vanilloid 1-mediated human lung epithelial cell toxicity.

Activation of intracellular transient receptor potential vanilloid-1 (TRPV1) in human lung cells causes endoplasmic reticulum (ER) stress, increased expression of proapoptotic GADD153 (growth arrest- and DNA damage-inducible transcript 3), and cytotoxicity. However, in cells with low TRPV1 expression, cell death is not inhibited by TRPV1 antagonists, despite preventing GADD153 induction. In this study, chemical variants of the capsaicin analog nonivamide were synthesized and used to probe the relationship between TRPV1 receptor binding, ER calcium release, GADD153 expression, and cell death in TRPV1-overexpressing BEAS-2B, normal BEAS-2B, and primary normal human bronchial epithelial lung cells.

Improved cytochrome P450 3A4 molecular models accurately predict the Phe215 requirement for raloxifene dehydrogenation selectivity.

The use of molecular modeling in conjunction with site-directed mutagenesis has been extensively used to study substrate orientation within cytochrome P450 active sites and to identify potential residues involved in the positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies is strongly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (Protein Data Bank entry 1W0E) to predict the sites of metabolism of the known CYP3A4 substrate raloxifene.

Potent mutagenicity of 3-methylindole requires pulmonary cytochrome P450-mediated bioactivation: a comparison to the prototype cigarette smoke mutagens B(a)P and NNK.

3-Methylindole (3MI) is a preferential pneumotoxicant found in cigarette smoke. A number of lung-expressed human cytochrome P450 enzymes, including 1A1, 2F1, and 2A13, catalyze the metabolism of 3MI to reactive intermediates that fragment DNA, measured with the Comet assay to assess DNA damage, in a cytochrome P450-dependent manner in primary normal human lung cells in culture, but the mutagenesis of 3MI has been controversial. In the present study, the mutagenic potential of 3MI was compared to the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

The inhaled glucocorticoid fluticasone propionate efficiently inactivates cytochrome P450 3A5, a predominant lung P450 enzyme.

Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs.

CYP3A4-Mediated oxygenation versus dehydrogenation of raloxifene.

Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein.

3-Methylindole metabolites induce lung CYP1A1 and CYP2F1 enzymes by AhR and non-AhR mechanisms, respectively.

3-Methylindole (3MI) is a highly selective pneumotoxicant that is present in abundant amounts (as high as 1.4 mug/cigarette) in cigarette smoke. Several human cytochrome P450 enzymes that are expressed in lung, such as CYP1A1, CYP2F1, CYP2A13, and CYP4B1, catalyze the dehydrogenation of 3MI to the reactive intermediate 3-methyleneindolenine, which alkylates DNA and induces cell death through apoptosis.

3-Methylindole is mutagenic and a possible pulmonary carcinogen.

Previous work has shown that bioactivation of the cigarette smoke pneumotoxicant 3-methylindole (3MI) by pulmonary cytochrome P450 enzymes is directly associated with formation of DNA adducts. Here, we present evidence that normal human lung epithelial cells, exposed to low micromolar concentrations of 3MI, showed extensive DNA damage, as measured by the comet assay, with similar potency to the prototypical genotoxic agents, doxorubicin and irinotecan. The DNA damage caused by 3MI was predominantly caused by single-strand breaks.

The pneumotoxin 3-methylindole is a substrate and a mechanism-based inactivator of CYP2A13, a human cytochrome P450 enzyme preferentially expressed in the respiratory tract.

3-Methylindole (3MI), a respiratory tract toxicant, can be metabolized by a number of cytochromes P450 (P450), primarily through either dehydrogenation or epoxidation of the indole. In the present study, we assessed the bioactivation of 3MI by recombinant CYP2A13, a human P450 predominantly expressed in the respiratory tract. Four metabolites were detected, and the two principal ones were identified as indole-3-carbinol (I-3-C) and 3-methyloxindole (MOI).

Dehydrogenation of the indoline-containing drug 4-chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (indapamide) by CYP3A4: correlation with in silico predictions.

4-Chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (indapamide), an indoline-containing diuretic drug, has recently been evaluated in a large Phase III clinical trial (ADVANCE) with a fixed-dose combination of an angiotensin-converting enzyme inhibitor, perindopril, and shown to significantly reduce the risks of major vascular toxicities in people with type 2 diabetes. The original metabolic studies of indapamide reported that the indoline functional group was aromatized to indole through a dehydrogenation pathway by cytochromes P450. However, the enzymatic efficiency of indapamide dehydrogenation was not elucidated.

A novel method to detect unlabeled inorganic nanoparticles and submicron particles in tissue by sedimentation field-flow fractionation.

Unlabelled: A novel methodology to detect unlabeled inorganic nanoparticles was experimentally demonstrated using a mixture of nano-sized (70 nm) and submicron (250 nm) silicon dioxide particles added to mammalian tissue. The size and concentration of environmentally relevant inorganic particles in a tissue sample can be determined by a procedure consisting of matrix digestion, particle recovery by centrifugation, size separation by sedimentation field-flow fractionation (SdFFF), and detection by light scattering.

Background: Laboratory nanoparticles that have been labeled by fluorescence, radioactivity, or rare elements have provided important information regarding nanoparticle uptake and translocation, but most nanomaterials that are commercially produced for industrial and consumer applications do not contain a specific label.

Single mutations change CYP2F3 from a dehydrogenase of 3-methylindole to an oxygenase.

Pulmonary cytochrome P450 2F3 (CYP2F3) catalyzes the dehydrogenation of the pneumotoxin 3-methylindole (3MI) to an electrophilic intermediate, 3-methyleneindolenine, which is responsible for the toxicity of the parent compound. Members of the CYP2F subfamily are the only enzymes known to exclusively dehydrogenate 3MI, without detectable formation of oxygenation products. Thus, CYP2F3 is an attractive model to study dehydrogenation mechanisms.