Christopher A. Reilly

Development and validation of an alternative dermal sensitization test: The mouse ear swelling test (MEST)

Traditional predictive tests for dermal sensitization in humans use the albino guinea pig as a model. A number of factors make the prospect of an alternative attractive. Guinea pig designs are labor intensive, require significant animal, caging, and husbandry resources, and are expensive. Extensive development and validation was conducted of an alternative using swelling of mouse ears as a quantitative end point. Ten strains of mice, ten age groups, both sexes, induction forms (number, route, timing), the use of an adjuvant, different vehicles and intervals to challenge, two induction sites, and three measurement intervals were evaluated. A methodology was developed for preparing induction sites to increase test sensitivity. A small battery of standard compounds was used to evaluate these design variables and a final test design was developed. The basic process was also demonstrated to occur in rats and guinea pigs and to be dose responsive. The final mouse ear swelling test (MEST) design was used to evaluate 72 materials representing a broad spectrum of chemicals and testing problems. These included 49 known positives and 23 known negatives. Guinea pig maximization test data on 37 of these resulting by studies conducted in our laboratories, along with closed patch guinea pig and human test data on many of these compounds, are also reported here for the first time. The MEST correctly identified 71 of 72 materials as potential human sensitizers or nonsensitizers. Additionally, both the efficacy of an occluded patch induction method and the duration of responsiveness of mice were evaluated. In the studies, the MEST was found to be an accurate, sensitive, and efficient alternative test design for evaluating delayed-contact sensitization.

A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda

Porphyria cutanea tarda (PCT), the most common form of porphyria in humans, is due to reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Previous studies have demonstrated that protein levels of URO-D do not change when catalytic activity is reduced, suggesting that an inhibitor of URO-D is generated in hepatocytes. Here, we describe the identification and characterization of an inhibitor of URO-D in liver cytosolic extracts from two murine models of PCT: wild-type mice treated with iron, δ-aminolevulinic acid, and polychlorinated biphenyls; and mice with one null allele of Uro-d and two null alleles of the hemochromatosis gene (Uro-d+/−, Hfe−/−) that develop PCT with no treatments. In both models, we identified an inhibitor of recombinant human URO-D (rhURO-D). The inhibitor was characterized by solid-phase extraction, chromatography, UV-visible spectroscopy, and mass spectroscopy and proved to be uroporphomethene, a compound in which one bridge carbon in the uroporphyrinogen macrocycle is oxidized. We synthesized uroporphomethene by photooxidation of enzymatically generated uroporphyrinogen I or III. Both uroporphomethenes inhibited rhURO-D, but the III isomer porphomethene was a more potent inhibitor. Finally, we detected an inhibitor of rhURO-D in cytosolic extracts of liver biopsy samples of patients with PCT. These studies define the mechanism underlying clinical expression of the PCT phenotype, namely oxidation of uroporphyrinogen to uroporphomethene, a competitive inhibitor of URO-D. The oxidation reaction is iron-dependent.

Quantitative analysis of capsaicinoids in fresh peppers, oleoresin capsicum and pepper spray products.

Liquid chromatography-mass spectrometry was used to identify and quantify the predominant capsaicinoid analogues in extracts of fresh peppers, in oleoresin capsicum, and pepper sprays. The concentration of capsaicinoids in fresh peppers was variable. Variability was dependent upon the relative pungency of the pepper type and geographical origin of the pepper. Nonivamide was conclusively identified in the extracts of fresh peppers, despite numerous reports that nonivamide was not a natural product. In the oleoresin capsicum samples, the pungency was proportional to the total concentration of capsaicinoids and was related by a factor of approximately 15,000 Scoville Heat Units (SHU)/microg of total capsaicinoids. The principle analogues detected in oleoresin capsicum were capsaicin and dihydrocapsaicin and appeared to be the analogues primarily responsible for the pungency of the sample. The analysis of selected samples of commercially available pepper spray products also demonstrated variability in the capsaicinoid concentrations. Variability was observed among products obtained from different manufacturers as well as from different product lots from the same manufacturer. These data indicate that commercial pepper products are not standardized for capsaicinoid content even though they are classified by SHU. Variability in the capsaicinoid concentrations in oleoresin capsicum-based self-defense weapons could alter potency and ultimately jeopardize the safety and health of users and assailants.

Human Lung Epithelial Cells Express a Functional Cold-Sensing TRPM8 Variant

Several transient receptor potential (TRP) ion channels sense and respond to changes in ambient temperature. Chemical agonists of TRP channels, including menthol and capsaicin, also elicit sensations of temperature change. TRPM8 is a cold- and menthol-sensing ion channel that converts thermal and chemical stimuli into neuronal signals and sensations of cooling/cold. However, the expression and function of TRPM8 receptors in non-neuronal cells and tissues is a relatively unexplored area. Results presented here document the expression and function of a truncated TRPM8 variant in human bronchial epithelial cells. Expression of the TRPM8 variant was demonstrated by RT-PCR, cloning, and immunohistology. Receptor function was characterized using the prototypical TRPM8 agonist, menthol, and exposure of cells to reduced temperature (18 degrees C). The TRPM8 variant was expressed primarily within endoplasmic reticulum membranes of lung epithelial cells and its activation was attenuated by thapsigargin, the cell-permeable TRPM8 antagonist N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide, and shRNA-induced suppression of TRPM8 expression. Activation of the TRPM8 variant in lung cells was coupled with enhanced expression of the inflammatory cytokines IL-6 and IL-8. Collectively, our results suggest that this novel TRPM8 variant receptor may function as a modulator of respiratory physiology caused by cold air, and may partially explain asthmatic respiratory hypersensitivity to cold air.

Transient Receptor Potential Vanilloid 1 Agonists Cause Endoplasmic Reticulum Stress and Cell Death in Human Lung Cells

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-selective ion channel expressed in human lung cells. We show that activation of the intracellular subpopulation of TRPV1 causes endoplasmic reticulum (ER) stress and cell death in human bronchial epithelial and alveolar cells. TRPV1 agonist (nonivamide) treatment caused calcium release from the ER and altered the transcription of growth arrest- and DNA damage-inducible transcript 3 (GADD153), GADD45α, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist N-(4-tert-butylbenzyl)-N′-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea (LJO-328) inhibited mRNA responses and cytotoxicity. EGTA and ruthenium red inhibited cell surface TRPV1 activity, but they did not prevent ER stress gene responses or cytotoxicity. Cytotoxicity paralleled eukaryotic translation initiation factor 2, subunit 1 (EIF2α) phosphorylation and the induction of GADD153 mRNA and protein. Transient overexpression of GADD153 caused cell death independent of agonist treatment, and cells selected for stable overexpression of a GADD153 dominant-negative mutant exhibited reduced sensitivity. Salubrinal, an inhibitor of ER stress-induced cytotoxicity via the EIF2αK3/EIF2α pathway, or stable overexpression of the EIF2α-S52A dominant-negative mutant also inhibited cell death. Treatment of the TRPV1-null human embryonic kidney 293 cell line with TRPV1 agonists did not initiate ER stress responses. Likewise, n-benzylnonanamide, an inactive analog of nonivamide, failed to cause ER calcium release, an increase in GADD153 expression, and cytotoxicity. We conclude that activation of ER-bound TRPV1 and stimulation of GADD153 expression via the EIF2αK3/EIF2α pathway represents a common mechanism for cytotoxicity by cell-permeable TRPV1 agonists. These findings are significant within the context of lung inflammatory diseases where elevated concentrations of endogenous TRPV1 agonists are probably produced in sufficient quantities to cause TRPV1 activation and lung cell death.

Determination of capsaicin, dihydrocapsaicin, and nonivamide in self-defense weapons by liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the analysis of capsaicin, dihydrocapsaicin, and nonivamide in pepper spray products have been developed. Chromatographic separation of the capsaicinoid analogues was achieved using a reversed-phase HPLC column and a stepwise gradient of methanol and distilled water containing 0.1% (v/v) formic acid. Identification and quantification of the capsaicinoids was achieved by electrospray ionization single-stage mass spectrometry monitoring the protonated molecules of the internal standard (m/z 280), capsaicin (m/z 306), dihydrocapsaicin (m/z 308), and nonivamide (m/z 294) or by tandem mass spectrometry monitoring the appropriate precursor-to-product-ion transitions. The plot of concentration versus peak area ratio was linear over the range of 10-750 ng/ml using LC-MS and 10-500 ng/ml using LC-MS-MS. However, to accurately quantify the capsaicinoids in the pepper spray products calibration curves between 10 and 1000 ng were constructed and fit using a weighted quadratic equation. Using the quadratic curve, the accuracy of the assay ranged from 91 to 102% for all analytes. The intra-assay precision (RSD) for capsaicin was 2% at 25 ng/ml, 10% at 500 ng/ml, and 3% at 800 ng/ml. The inter-assay precision (RSD) for capsaicin was 6% at 25 ng/ml, 6% at 500 ng/ml, and 9% at 800 ng/ml. Similar values for inter- and intra-assay precision were experimentally obtained for both dihydrocapsaicin and nonivamide. The analysis of selected pepper spray products demonstrated that the capsaicinoid concentration in the products ranged from 0.7 to 40.5 microg/microl.

Metabolism of capsaicinoids by P450 enzymes: a review of recent findings on reaction mechanisms, bio-activation, and detoxification processes

Capsaicinoids are botanical irritants present in chili peppers. Chili pepper extracts and capsaicinoids are common dietary constituents and important pharmaceutical agents. Use of these substances in modern consumer products and medicinal preparations occurs worldwide. Capsaicinoids are the principals of pepper spray self-defense weapons and several over-the-counter pain treatments as well as the active component of many dietary supplements. Capsaicinoids interact with the capsaicin receptor (a.k.a., VR1 or TRPV1) to produce acute pain and cough as well as long-term analgesia. Capsaicinoids are also toxic to many cells via TRPV1-dependent and independent mechanisms. Chemical modifications to capsaicinoids by P450 enzymes decreases their potency at TRPV1 and reduces the pharmacological and toxicological phenomena associated with TRPV1 stimulation. Metabolism of capsaicinoids by P450 enzymes also produces reactive electrophiles capable of modifying biological macromolecules. This review highlights data describing specific mechanisms by which P450 enzymes convert the capsaicinoids to novel products and explores the relationship between capsaicinoid metabolism and its effects on capsaicinoid pharmacology and toxicology.

Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures

Recognition of temperature is a critical element of sensory perception and allows mammals to evaluate both their external environment and internal status. The respiratory epithelium is constantly exposed to the external environment, and prolonged inhalation of cold air is detrimental to human airways. However, the mechanisms responsible for adverse effects elicited by cold air on the human airways are poorly understood. Transient receptor potential melastatin family member 8 (TRPM8) is a well-established cold- and menthol-sensing cation channel. We recently discovered a functional cold- and menthol-sensing variant of the TRPM8 ion channel in human lung epithelial cells. The present study explores the hypothesis that this TRPM8 variant mediates airway cell inflammatory responses elicited by cold air/temperatures. Here, we show that activation of the TRPM8 variant in human lung epithelial cells leads to increased expression of several cytokine and chemokine genes, including IL-1alpha, -1beta, -4, -6, -8, and -13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-alpha. Our results provide new insights into mechanisms that potentially control airway inflammation due to inhalation of cold air and suggest a possible role for the TRPM8 variant in the pathophysiology of asthma.

Measurement of Lipid Peroxidation

There is currently considerable interest in what is termed “oxidative stress,” or the oxidation of biological macromolecules, with emphasis on its involvement in various diseases and toxicities and methods to limit either its occurrence or effects. This unit describes traditional methods to measure the extent or rate of lipid peroxidations, including assays for conjugated dienes, lipid hydroperoxides, the polyunsaturated lipid breakdown product malondialdehyde, and hemolysis, along with discussion of alternative methods.

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. 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.