Reduction of Tetrathionate by Mammalian Thioredoxin Reductase

Tetrathionate, a polythionate oxidation product of microbial hydrogen sulfide and reactive oxygen species from immune cells in the gut, serves as a terminal electron acceptor to confer a growth advantage for Salmonella and other enterobacteria. Here we show that the rat liver selenoenzyme thioredoxin reductase (Txnrd1, TR1) efficiently reduces tetrathionate <i>in vitro</i>. Furthermore, lysates of selenium-supplemented murine macrophages also displayed activity toward tetrathionate, while cells lacking TR1 were unable to reduce tetrathionate. These studies suggest that upregulation of TR1 expression, via selenium supplementation, may modulate the gut microbiome, particularly during inflammation, by regulating the levels of tetrathionate

Targeting of Histone Acetyltransferase p300 by Cyclopentenone Prostaglandin Δ¹²-PGJ₂ through Covalent Binding to Cys¹⁴³⁸

Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys¹⁴³⁸ in the substrate binding site could be targeted by Δ¹²-prostaglandin J₂ (Δ¹²-PGJ₂), a cyclopentenone prostaglandin (CyPG) derived from PGD₂. We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,β-unsaturation in the cyclopentenone ring of Δ¹²-PGJ₂ was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-Δ^12,14-PGJ₂, which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ¹²-PGJ₂ places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys¹⁴³⁸ of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ¹²-PGJ₂ with Cys¹⁴³⁸. Using biotinylated derivatives of Δ¹²-PGJ₂ and 9,10-dihydro-15-deoxy-Δ^12,14-PGJ₂, we demonstrate the covalent interaction of Δ¹²-PGJ₂ with the p300 HAT domain, but not the latter. In agreement with the <i>in vitro</i> filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ¹²-PGJ₂ also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in <i>in vitro</i> assays. This study demonstrates, for the first time, that Δ¹²-PGJ₂ inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity

Evaluation of the Stability, Bioavailability, and Hypersensitivity of the Omega-3 Derived Anti-Leukemic Prostaglandin: Δ¹²-Prostaglandin J₃

<div><p>Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ¹²-PGJ₃, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in <i>in vitro</i> and <i>in vivo</i> models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ¹²-PGJ₃. The stability of Δ¹²-PGJ₃ was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ¹²-PGJ₃ in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ¹²-PGJ₃ being a downstream metabolite of PGD₃ was tested <i>in vitro</i> using primary mouse bone marrow-derived mast cells (BMMCs) and <i>in vivo</i> mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD₂ receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ¹²-PGJ₃ was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ¹²-PGJ₃ was bioavailable and well absorbed into systemic circulation with a <i>C_max</i> of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ¹²-PGJ₃ did not induce degranulation in BMMCs nor increase histamine. In addition, <i>in vivo</i> testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ¹²-PGJ₃ and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ¹²-PGJ₃ was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ¹²-PGJ₃ failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ¹²-PGJ₃ to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.</p></div

<i>In vitro</i> effect of Δ¹²-PGJ₃ and ZK118182 on BMMCs.

<p>(A) Cytological evaluation for drug hypersensitivity upon treatment of BMMCs with Δ¹²-PGJ₃ and ZK118182. Cultured BMMCs (1×10⁶ cells/mL) were treated with Δ¹²-PGJ₃ and ZK118182 at 0.1 µM for 12 h, followed by fixation and staining with toluidine blue. BMMCs treated with ionomycin (1 µM) for 1 h was used as a positive control. All treatments were carried out in triplicate and a representative image is shown (magnification: 32X). (B) The cells and media treated as mentioned above were used for the estimation of histamine (total and released) using EIA method. (C–D) Real time PCR analysis was carried-out for pro-inflammatory gene markers upon treatment of BMMCs with PBS, ΔΔ¹²-PGJ₃ and ZK118182 as mentioned above (C-6 h; D-12 h). The data shown are mean ± SEM (n = 3) and statistical significance are represented as *- <i>p</i>≤0.05; **- <i>p</i>≤0.01; ***- <i>p</i>≤0.001 respectively.</p

Calculated half-life (t_1/2) and rate constant (k) for Δ¹²-PGJ₃^#.

#<p>Half-life (t_1/2) and Reaction rate constant (k) (indicated in square brackets) were calculated based on Arrhenius equation-first order kinetics for Δ¹²-PGJ₃ at various incubated temperatures and simulated physiological conditions.</p

<i>In vivo</i> evaluation for hypersensitivity by acute toxicity test.

<p>(A) Δ¹²-PGJ₃ (0.025 mg/kg body weight) was injected intraperitoneally into C57BL/6 mice. Post 6 h and 12 h the mice were sacrificed and the lungs extracted, fixed and stained by H&E. Mice treated with PBS for 12 h was used as a placebo control. All treatments were carried out in triplicates and a representative image has been shown (magnification: 10X). (B) Histamine release assay was carried out on the plasma from the mice treated as mentioned above by EIA method. (C) A real-time PCR analysis was carried out for pro-inflammatory gene markers upon treatment of mice with PBS and Δ¹²-PGJ₃ as mentioned above. The data shown are mean ± SEM (n = 3) and statistical significance are represented as *- <i>p</i>≤0.05.</p

<i>In vivo</i> evaluation for hypersensitivity by chronic toxicity test.

<p>(A) Δ¹²-PGJ₃ (0.025 and 0.05 mg/kg body weight) and ZK118182 (0.025 and 0.05 mg/kg body weight) were injected intraperitoneally into C57BL/6 mice. Post two weeks of treatment, the alveolar tissue was extracted, fixed, and stained with H&E. Mice treated with PBS were used as placebo control. All treatments were carried out in triplicates and a representative image has been shown (magnification: 10X). (B) Histamine release assay was carried out on the plasma from the mice treated as mentioned above by EIA method. (C–G) Multi-array Th1/Th2 cytokine analysis was carried-out using the plasma, upon treatment of mice as mentioned above. The figures shown are for: (C) IFN-γ, (D) TNF-α, (E) IL-10, (F) IL-12 and (G) mKC (CXCL-1). The data shown are mean ± SEM (n = 3) and statistical significance are represented as *- <i>p</i>≤0.05; **- <i>p</i>≤0.01; ***- <i>p</i>≤0.001 respectively.</p

Airways hyperresponsiveness test.

<p>Δ¹²-PGJ₃ and ZK118182 was injected intraperitoneally into C57BL/6 mice at (A) 0.025 mg/kg body weight/day and (B) 0.050 mg/kg body weight/day. Post seven days of administration, the AHR response was determined upon methacholine challenge. (C) Real-time PCR analysis was carried-out for expression of Th2 cytokines in the alveolar tissue of mice that were subjected to AHR analysis. The data shown here are mean ± SEM (n = 3) and statistical significance were calculated in comparison to (PBS) control and represented as *(<i>p</i>≤0.05).</p

Study on stability and bioavailability of Δ¹²-PGJ₃.

<p>(A–C) Δ¹²-PGJ₃ (0.5 µg/mL) was incubated in artificial gastric and intestinal juice (with or without pancreatin) for various time intervals and temperatures (4°C (A), 25°C (B), 37°C (C) and estimated for % of Δ¹²-PGJ₃ remaining by HPLC. Significant differences between concentrations Δ¹²-PGJ₃ at different time points as compared to t = 0 are indicated by *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001 respectively. Data shown are the mean ± SEM (n = 3). (D) Time course analysis for bioavailability of Δ¹²-PGJ₃ in mouse plasma. Δ¹²-PGJ₃ (0.025 mg/kg body weight) was injected intraperitoneally into C57BL/6 mice. The concentration of Δ¹²-PGJ₃ in plasma was measured at indicated times post injection by LC-MS/MS. The chemical structure of Δ¹²-PGJ₃ is shown in the inset of panel B. All data shown are mean ± SEM (n = 3) and statistical significance represented as *- <i>p</i>≤0.05; **- <i>p</i>≤0.01; ***- <i>p</i>≤0.001 respectively.</p