Garold S. Yost

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. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and ∆⁶-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of ∆⁶-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis.

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. Quantification of TRPA1 activation by 4 model PM revealed that diesel exhaust PM (DEP) and coal fly ash PM (CFA1) were TRPA1 agonists at concentrations >0.077 mg/mL. DEP was more potent, and approximately 97% of the activity of DEP was recovered by serial extraction of the solid DEP with ethanol and hexane/n-butyl chloride. Modification of the electrophile/agonist binding sites on TRPA1 (C621, C641, C665, and K710) to non-nucleophilic residues reduced TRPA1 activation by DEP and abolished activation by DEP extracts as well as multiple individual electrophilic chemical components of DEP. However, responses to CFA1 and DEP solids were not affected by these mutations. Activity-guided fractionation of DEP and high resolution mass spectroscopy identified several new DEP-derived TRPA1 agonists, and activation of mouse dorsal root ganglion neurons demonstrated that TRPA1 is a primary target for DEP in a heterogeneous population of primary sensory nerves. It is concluded that TRPA1 is a specific target for electrophilic chemical components of DEP and proposed that activation of TRPA1 in the respiratory tract is likely to be an important mechanism for DEP pneumotoxicity.

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. In addition, partial charges were incorporated into the P450 heme moiety to investigate the effect of the modified CYP3A4 model on metabolite prediction with the ligand docking program Autodock. Dehydrogenation of raloxifene to an electrophilic diquinone methide intermediate has been linked to the potent inactivation of CYP3A4. Active site residues involved in the positioning and/or catalysis of raloxifene supporting dehydrogenation were identified with the two models, and site-directed mutagenesis studies were conducted to validate the models. The addition of partial charges to the heme moiety improved the accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation and facilitating the identification of substrate−active site residue interactions. On the basis of the improved model, the Phe215 residue was hypothesized to play an important role in orienting raloxifene for dehydrogenation through a combination of electrostatic and steric interactions. Substitution of this residue with glycine or glutamine significantly decreased dehydrogenation rates without concurrent changes in the rates of raloxifene oxygenation. Thus, the improved structural model predicted novel enzyme−substrate interactions that control the selective dehydrogenation of raloxifene to its protein-binding intermediate.

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). 3MI, B(a)P, and NNK were incubated with the Salmonella typhimurium strain TA98, which is known to detect the most common subtype of cigarette smoke-induced mutagenicity, frameshift mutations in DNA, and with Salmonella typhimurium strain TA100, which detects base pair substitution mutants, with five sources of P450-mediated bioactivation: rat liver S9, human lung microsomes, recombinant CYP2A13, purified CYP2F3, and recombinant CYP1A1. Only B(a)P was mutagenic in TA100, and it was bioactivated by human lung microsomes and rat liver S9 sources of P450s. However, with the TA98 strain, CYP1A1, CYP2A13, CYP2F3, and human lung microsomes bioactivated 3MI to highly mutagenic intermediates, whereas neither human nor rat liver S9 subcellular fractions formed mutagenic intermediates from 3MI. Quantitative Western blot analysis verified that all three respiratory enzymes were present in human lung microsomes in widely varying amounts. These results indicate that metabolism of 3MI by human lung-expressed cytochrome P450 enzymes but not hepatic P450s elicits equivalent or higher mutagenicity than the prototype cigarette smoke mutagens B(a)P and NNK and indicates that 3MI is a likely human pulmonary carcinogen.

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. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5, and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with K_I, k_inact, and partition ratio of 16 μM, 0.027 min⁻¹, and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 μM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs.

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. However, previous studies could not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, ¹⁸O-incorporation studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene. These studies established that 3′-hydroxyraloxifene is produced exclusively via CYP3A4-mediated oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical O₂-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.

3-Methylindole (3MI) is a highly selective pneumotoxicant that is present in abundant amounts (as high as 1.4 μg/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. In addition, 3MI potently damages DNA at low concentrations (observable at 0.1 μM). However, it seemed possible that 3MI could induce the levels of P450 enzymes, so transcription and translation of 1A1 and 2F1 genes were measured in primary normal human bronchial epithelial cells. In this study, 3MI-induced DNA damage at the 10 μM concentration was ameliorated when P450 turnover was inactivated with the cytochrome P450 suicide substrate inhibitor 1-aminobenzotriazole. Thus, the observed DNA damage was cytochrome P450-dependent. Quantitative real-time polymerase chain reaction analysis revealed both concentration- and time-dependent increases in CYP1A1 and CYP2F1 transcription by the same 3MI concentrations that damaged DNA. Aryl hydrocarbon receptor (AhR) activation lead to CYP1A1 induction. Treatment with 3MI in combination with the AhR antagonist α-naphthoflavone prevented 3MI-mediated CYP1A1 induction, indicating that the induction was AhR-dependent. Conversely, CYP2F1 induction did not appear to require activation of AhR. These intriguing findings show that not only is induction of 1A1 and 2F1 caused by 3MI metabolites, rather than 3MI itself, but transcriptional activation of these pulmonary genes occurs through disparate mechanisms. Thus, the induction process, and subsequent increased bioactivation of 3MI to toxic intermediates, is a facile process that might enhance the acute toxicity and/or mutagenicity of this chemical.

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. Furthermore, we show that this damage decreased with time, given a subtoxic concentration, with detectable DNA fragmentation peaking 4 h after exposure and diminishing to untreated levels within 24 h. Pretreatment with an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1), NU1025, nearly doubled the DNA damage produced by 5μM 3MI, implying that PARP1, which among other activities, functions to repair single-strand breaks in DNA, repaired at least some of the 3MI-induced DNA fragmentation. A key cellular response to DNA damage, phosphorylation, and nuclear localization of p53 was seen at subtoxic levels of 3MI exposure. 3MI was highly mutagenic, with essentially the same potency as the prototype carcinogen, benzo[a]pyrene, only when a lung-expressed CYP2F3 enzyme was used to dehydrogenate 3MI to its putative DNA-alkylating intermediate. Conversely, a rat liver S9 metabolic system did not bioactivate 3MI to its mutagenic intermediate(s). Concentrations higher than 25μM caused apoptosis, which became extensive at 100μM, similar to the response seen with 10μM doxorubicin. Our findings indicate that there is a low concentration window in which 3MI can cause extensive DNA damage and mutation, without triggering apoptotic defenses, reinforcing the hypothesis that inhaled 3MI from cigarette smoke may be a potent lung-selective carcinogen.

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 consequence of indoline aromatization is that the product indoles might have dramatically different therapeutic potencies. Thus, studies that characterize dehydrogenation of the functional indoline of indapamide were needed. Here we identified several indapamide metabolic pathways in vitro with human liver microsomes and recombinant CYP3A4 that include the dehydrogenation of indapamide to its corresponding indole form, and also hydroxylation and epoxidation metabolites, as characterized by liquid chromatography/mass spectrometry. Indapamide dehydrogenation efficiency (V_max/K_m = 204 min/mM) by CYP3A4 was approximately 10-fold greater than that of indoline dehydrogenation. In silico molecular docking of indapamide into two CYP3A4 crystal structures, to evaluate the active site parameters that control dehydrogenation, produced conflicting results about the interactions of Arg212 with indapamide in the active site. These conflicting theories were addressed by functional studies with a CYP3A4R212A mutant enzyme, which showed that Arg212 does not seem to facilitate positioning of indapamide for dehydrogenation. However, the metabolites of indapamide were precisely consistent with in silico predictions of binding orientations using three diverse computer methods to predict drug metabolism pathways.

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. Methods Both nitric acid digestion and enzyme digestion were tested with liver and lung tissue as well as with cultured cells. Tissue processing with a mixture of protease enzymes is preferred because it is applicable to a wide range of particle compositions. Samples were visualized via fluorescence microscopy and transmission electron microscopy to validate the SdFFF results. We describe in detail the tissue preparation procedures and discuss method sensitivity compared to reported levels of nanoparticles in vivo. Conclusion Tissue digestion and SdFFF complement existing techniques by precisely identifying unlabeled metal oxide nanoparticles and unambiguously distinguishing nanoparticles (diameter<100 nm) from both soluble compounds and from larger particles of the same nominal elemental composition. This is an exciting capability that can facilitate epidemiological and toxicological research on natural and manufactured nanomaterials.