Phosphonated Trityl Probes for Concurrent in Vivo Tissue Oxygen and pH Monitoring Using Electron Paramagnetic Resonance-Based Techniques

Previously we proposed the concept of dual function pH and oxygen paramagnetic probes based on the incorporation of ionizable groups into the structure of persistent triarylmethyl radicals, TAMs (<i>J. Am. Chem. Soc.</i> <b>2007</b>, <i>129</i>, 7240–7241). In this paper, we synthesized an asymmetric monophosphonated TAM probe with the simplest doublet hfs pattern ideally suited for dual function electron paramagnetic resonance (EPR)-based applications. An extraordinary low line width of the synthesized deuterated derivative, p₁TAM-D (Δ<i>H</i>_pp ≤ 50 mG, Lorentz line width, ≤20 mG) results in high sensitivity to <i>p</i>O₂ due to oxygen-induced line broadening (ΔLW/Δ<i>p</i>O₂ ≈ 0.5 mG/mmHg or ≈400 mG/mM); accuracy of <i>p</i>O₂ measurement, ≈1 mmHg). The presence of a phosphono group in the p₁TAM-D structure provides pH sensitivity to its EPR spectra in the physiological range of pH from 5.9 to 8.2 with the ratio of signal intensities of protonated and deprotonated states being a reliable pH marker (accuracy of pH measurements, ± 0.05). The independent character of pH and [O₂] effects on the EPR spectra of p₁TAM-D provides dual functionality to this probe. The L-band EPR studies performed in breast tumor-bearing mice show a significant difference in extracellular pH and <i>p</i>O₂ between tumor and normal mammary gland tissues, as well as the effect of animal breathing with 100% O₂ on tissue oxygenation. The developed dual function phosphonated p₁TAM-D probe provides a unique tool for in vivo concurrent tissue oxygen and pH monitoring

RST increases erythroid precursors in bone marrow, rescued by glucocorticoid receptor antagonism.

<p>Wildtype female C57Bl/6 mice were left unstressed (Control), subjected to 6 hrs of restrain stress (RST), subjected to RST and treated daily with 0.4 mg/day RU486 in 50 µL of 50% ethanol/50% PBS (RST + RU486), or subjected to RST and treated daily with 50 µL of 50% ethanol/50% PBS (RST + Vehicle). RU486 and vehicle treatments were given by subcutaneous injection at 0900h immediately before RST exposure. After 21 days and immediately following sacrifice, bone marrow was collected from femurs and immunostained with antibodies describing megakaryocyte-erythroid progenitor (MEP) cells (lin, CD34, CD16/32, Sca-1, and c-kit). The percent lin-/CD34-/CD16/32low/Sca-1-/c-kit+ MEP subpopulation relative to total cells were determined by flow cytometry. Mice subjected to RST had a significantly higher percentage of MEPs than unstressed control mice while RU486 suppressed elevation of MEPs in RST mice. For each data point, n = 5. Data shown is mean +SEM. *, p < 0.05 by ANOVA. </p

RST increases circulating reticulocytes.

<p>A cohort of wildtype female C57Bl/6 mice were left unstressed (Control) or subjected to 6 hrs of restraint stress (RST) for 28 days followed by humane sacrifice and tissue collection. Another cohort of mice was allowed to recover for an additional 14 days without RST (Post-stress) prior to sacrifice and tissue collection. Whole blood was collected via retro-orbital bleeds and circulating reticulocytes quantified during (days 0-28) and following the stress period (days 29-42) using a clinical flow hemocytometer. Data were collected without repeated sampling of individuals. Mice subjected to RST presented significantly higher numbers of reticulocytes compared to unstressed control mice. For each data point n = 9-15 individuals. Data shown is mean +SEM. *, p < .05 by ANOVA vs. unstressed control mice.</p

RST elevates erythropoiesis in primary and secondary tissues.

<p>From the same experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077935#pone-0077935-g002" target="_blank">Figure 2</a>, tissue was collected from control mice or mice exposed to RST after 21 days. Cells from bone marrow and homogenized spleens and livers were immunostained with fluorochrome-conjugated antibodies for erythrocyte differentiation (CD71 and Ter119) and quantified by flow cytometry as a percentage of lineage negative cells in primary (Bone marrow) and secondary (Spleen) and (Liver) hematopoietic tissues. RST increased CD71+/Ter119+ cell populations from these tissues compared to tissue from unstressed mice. Data were collected without repeated sampling of individuals. For each data point, n = 5. Data shown is mean +SEM. *, p < 0.05 by Student T-test vs. unstressed control mice in same tissue.</p

RST increases expression of <i>KLF1</i> in MEP cells, rescued by glucocorticoid receptor antagonism.

<p>Wildtype female C57Bl/6 mice were left unstressed (Control), subjected to 6 hrs of restrain stress (RST), subjected to RST and treated daily with 0.4 mg/day RU486 in 50 µL of 50% ethanol/50% PBS (RST + RU486), or subjected to RST and treated daily with 50 µL of 50% ethanol/50% PBS (RST + Vehicle). RU486 and vehicle treatments were given by subcutaneous injection at 0900h immediately before RST exposure. After 21 days and immediately following sacrifice, bone marrow was collected from femurs and immunostained with antibodies for lin, CD34, CD16/32, Sca-1, and c-kit. The lin-/CD34-/CD16/32low/Sca-1-/c-kit+ MEP subpopulation were collected by flow-assisted cell sorting. Cells were subjected to Trizol, RNA isolated and cDNA synthesized. Expression of the erythropoietic transcription factors (<i>KLF-1</i>, <i>c-myb</i>, <i>Fli1</i>, <i>Runx1</i>, <i>c-kit</i>, <i>Gata1</i>) and glucocorticoid receptor (GR) were examined by Real-time PCR. mRNA expression for all four mouse groups is expressed as fold change relative to the control group. Of all genes investigated, only <i>KLF-1</i> was significantly increased by RST. RU486 treatment abrogated RST-induced <i>KLF-1</i> levels similar to unstressed control mice. For each data point, n = 7-13. Data shown is mean +SEM. *, p < 0.05 by ANOVA for each mRNA in all four mouse groups.</p

The glucocorticoid receptor antagonist RU486 does not prevent biological stress response.

<p>A) Wildtype female C57Bl/6 mice were left unstressed (Control), subjected to 6 hrs of restrain stress (RST), subjected to RST and treated daily with 0.4 mg/day RU486 in 50 µL of 50% ethanol/50% PBS (RST + RU486), or subjected to RST and treated daily with 50 µL of 50% ethanol/50% PBS (RST + Vehicle). RU486 and vehicle treatments were given by subcutaneous injection at 0900h immediately before RST exposure. After 21 days, the mice were sacrificed and whole blood collected via atrial puncture. Serum corticosterone levels were determined using a ¹²⁵I double antibody radioimmunoassay. Serum corticosterone was significantly elevated in all conditions compared to unstressed control mice. For each data point n = 5-8 individuals. Data shown is mean +SEM. *, p < .05 by ANOVA. B) After whole blood collection, total mouse body weight was recorded. Spleen, thymus, and adrenal glands were subsequently collected, weighed, and standardized as a percent of total body weight. Mice subjected to RST, with or without RU486 or vehicle treatment, weighed significantly less than unstressed control mice. The spleens and thymuses from the mice subjected to RST, with or without RU486 or vehicle treatment, were significantly lighter than control mice. In contrast, adrenal glands from mice subjected to RST were significantly heavier than control mice and RU486 or vehicle treatment had no effect. For each data point n = 5-8 individuals. Data shown is mean +SEM. *, p < .05 by ANOVA.</p

Microarray analysis predicts disrupted hematopoiesis in response to RST.

<p>Following seven days of RST, mice were sacrificed and total mRNA was extracted from the whole blood using and purified over solid phase silica columns. mRNA with integrity scores >8.0 was reverse-transcribed and run on Mouse Genome 430 2.0 Affymetrix GeneChips® (control, n = 4 and RST, n = 4). Following internal corrections, genes with significance levels of p < 0.05 and greater than 1.5-fold change were uploaded with corresponding Genbank accession numbers to Ingenuity Pathways Analysis functional network analysis. (<b>A</b>) Hierarchical clustering was performed on genes demonstrating statistically significant changes, using Partek Genomic Suite, the Euclidean algorithm with average linkage, and standardized gene expression. (<b>B</b>) Ingenuity Pathways Analysis suggest significant disruption of hematopoiesis and hematological function in response to RST. (<b>C</b>) Pathway mapping indicates elevated expression of the pro-erythroid transcription factor KLF1 in response to RST and that this change could be affected through the glucocorticoid receptor (NR3C1) In (<b>A</b>) and (<b>C</b>), green represents down-regulation and red up-regulation of respective gene expression. </p

RST does not affect erythropoietin levels.

<p>Wildtype female C57Bl/6 mice were left unstressed (Control), subjected to 6 hrs of restrain stress (RST), subjected to RST and treated daily with 0.4 mg/day RU486 in 50 µL of 50% ethanol/50% PBS (RST + RU486), or subjected to RST and treated daily with 50 µL of 50% ethanol/50% PBS (RST + Vehicle). RU486 and vehicle treatments were given by subcutaneous injection at 0900h immediately before RST exposure. After 21 days, whole blood was collected by atrial puncture and serum levels of erythropoietin were quantified by ELISA. There was no statistical difference between mouse groups. For each data point, n = 5. Data shown is mean +SEM by ANOVA.</p

Experiments and simulations of pH in tumors with wild-type, HIF-1<i>α</i>-, and HIF-2<i>α</i>-deficient macrophages (WT, HIF-1<i>α</i> KO, and HIF-2<i>α</i> KO).

<p>Horizontal axis represents time (in days) and vertical axis shows the pH value. (a): Experimental data of pH against time with error bars. Red: WT; Blue: HIF-1<i>α</i> KO; Green: HIF-2<i>α</i> KO. (b) – (d): Comparison of experiments (dots with error bars) and numerical simulations (dash curves) of pH in tumor with WT, HIF1-<i>α</i>, and HIF-2<i>α</i> KO macrophages, respectively.</p

Profiles of the functions , , and .

<p>Thresholds of necroxia, hypoxia and normoxia are marked as , and , respectively.</p