Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic MUC5AC Expression by Vasaka (Adhatoda vasica; Malabar Nut) Tea.

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.

Analysis of the nutritional supplement 1AD, its metabolites, and related endogenous hormones in biological matrices using liquid chromatography-tandem mass spectrometry.

1,5alpha-Androsten-3beta,17beta-diol and/or 1,5alpha-androsten-3,17-dione (1AD) is an over-the-counter pro-hormone nutritional supplement designed to enhance strength and performance in athletes. 1AD purportedly mimics the pharmacological activity of testosterone through activation of the pro-hormones to their active form 1,5alpha-androsten-17beta-ol-3-one or Delta(1)-testosterone. This testosterone analogue ostensibly possesses strong androgenic potency without the adverse effects associated with aromatization of testosterone to estrogens. We have developed a highly sensitive and selective liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of 1AD, its analogues, and several structurally related endogenous hormones in plasma and urine. The limits of quantitation for the analytes ranged from 0.25 to 0.5 ng/mL. The accuracy of the assay was 92-113% with a precision of 2-12% relative standard deviation (RSD) for all analytes at 1.0, 5.0, and 15.0 ng/mL, respectively. The interassay precision was 6-16% RSD, and the accuracy was 90-105%. We have used this assay to determine the unconjugated and total (conjugated and unconjugated) concentrations of 1AD, its analogues, androstenediol, androstenedione, testosterone, dihydrotestosterone, and estradiol, in plasma and urine, as well as to investigate the metabolic fate of the three 1AD analogues (diol, dione, and active forms) when incubated with rat liver microsomes or rat testicular homogenates. Concentrations of both unconjugated and total testosterone in plasma were approximately 1.5 ng/mL and ranged from undetectable to 4.1 ng/mL in urine. 1AD and its analogues were not detected in plasma or urine. In vitro metabolism experiments using rat liver microsomes and testicular homogenates provided evidence for the interconversion of the three 1AD analogues, biosynthesis, and decomposition of several endogenous hormones, as well as evidence for 1AD analogue-induced changes in the typical profiles of testosterone and androstenedione in testicular tissue.

Metabolism of capsaicin by cytochrome P450 produces novel dehydrogenated metabolites and decreases cytotoxicity to lung and liver cells.

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and omega-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).