sj-docx-1-imj-10.1177_10815589221145043 – Supplemental material for Organochlorine pesticides, oxidative stress biomarkers, and leukemia: a case–control study

Supplemental material, sj-docx-1-imj-10.1177_10815589221145043 for Organochlorine pesticides, oxidative stress biomarkers, and leukemia: a case–control study by Arash Rafeeinia, Gholamreza Asadikaram, Mehrnaz Karimi Darabi, Moslem Abolhassani, Vahid Moazed and Mojtaba Abbasi-Jorjandi in Journal of Investigative Medicine</p

The L-arginine/L-ornithine antiporter ArcD2 is active in liposomes with anionic lipids but not in vesicles that do not contain lipids or surfactants with anionic headgroups.

<p>ArcD2 was reconstituted in liposomes and niosomes at 1 to 400 protein to lipid ratio (w/w). A: Schematic representation of the transport reaction. B: ArcD activity was measured using radiolabeled arginine. Green lines: niosomes composed of unsaturated surfactants plus cholesterol; blue lines: liposomes composed of unsaturated lipids plus cholesterol; black lines: liposomes composed of unsaturated lipids of which 38% is anionic (phosphatidylglycerol). Representative traces of one out of three independent experiments are shown. C: Incorporation of ArcD2 into vesicles was confirmed by Western blot analysis. 1: solubilized ArcD2, purified protein before reconstitution; 2: Unsaturated surfactants + cholesterol; 3: Saturated surfactants + cholesterol; 4: Unsaturated lipids + cholesterol.</p

Lipid composition of niosomes and liposomes used in this study.

<p>Lipid composition of niosomes and liposomes used in this study.</p

Filter-extruded niosomes decrease in size upon freezing and thawing.

<p>A: Size of vesicles composed of unsaturated surfactants plus cholesterol (green: without freezing and thawing; red: with freezing and thawing), and vesicles composed of unsaturated lipids plus cholesterol (black: without freezing and thawing; blue: with freezing and thawing), measured by dynamic light scattering. Prior to the analysis the vesicles were extruded 15 times through a 200 nm polycarbonate filter. B-C: Cryo-EM pictures of niosomes composed of unsaturated surfactants plus cholesterol without (B) and with five freeze and thaw cycles (C). Niosomes appear smaller due to the freezing and thawing steps. As guidance, all niosomes are indicated with a red arrow in the right picture. In contrast, cryo-EM pictures of liposomes composed of unsaturated lipids plus cholesterol without (D) and with five freeze and thaw cycles (E) appear similar in size but the degree of multilamellarity decreases by the freezing-thawing and subsequent extrusion step.</p

Membrane permeability of niosomes and liposomes.

<p>A: Graphical representation of the ion permeability of the vesicles. B: Proton permeability measured by fluorescence of the pH-sensitive dye pyranine in liposomes composed of unsaturated lipids plus cholesterol in the presence and absence of the sodium ionophore ETH-157. ETH-157 (5 μM, final concentration) or ethanol (0.1% v/v) were present from the start of the experiment. At time point 0 (indicated by an arrow), the medium pH was decreased from 7.5 to 6.3 by the addition of 10 mM HCl (large pulse) or from 7.5 to 7.0 by the addition of 4 mM HCl (small pulse). Black line: ethanol, small pulse; red line: ETH-157, small pulse; green line: ethanol, large pulse; blue line: ETH-157, large pulse. For comparison, liposomes composed of saturated lipids plus cholesterol subjected to a large HCl pulse (in the absence of ETH-157) are shown in grey. Average values of two experiments are shown. C: Proton permeability of niosomes composed of unsaturated surfactants plus cholesterol in the absence (0.1% v/v ethanol) or presence of the sodium ionophore ETH-157 (5 μM, final concentration). At time point 0 (indicated by an arrow), the medium pH was decreased from 7.5 to 6.3 by the addition of 10 mM HCl (large pulse) or from 7.5 to 7.0 by the addition of 4 mM HCl (small pulse). Black line: ethanol, small pulse; red line: ETH-157, small pulse; green line: ethanol, large pulse; blue line: ETH-157, large pulse. Niosomes composed of saturated surfactants plus cholesterol subjected to a large HCl pulse (in the absence of ETH-157) are shown in grey. Average values of two independent experiments are shown. D: KCl permeability of liposomes and niosomes filled with the fluorescent dye calcein (5 mM) after osmotic upshift by KCl. The arrow at 50s indicates the moment 0.4 M KCl (final concentration) was added. Green lines: niosomes composed of unsaturated surfactants plus cholesterol; red lines: niosomes composed of saturated surfactants plus cholesterol; black lines: liposomes composed of unsaturated lipids plus cholesterol; blue lines: liposomes composed of saturated lipids plus cholesterol. Representative traces of one out of three independent experiments are shown. E: Stability of liposomes and niosomes filled with the fluorescent dye calcein (5 mM) after osmotic upshift by glycerol. At 50s (indicated by a black arrow), 0.667 M glycerol was added (osmolarity comparable to that of 0.4M KCl); line color as indicated under B. Representative traces of one out of two independent experiments are shown. F: Stopped-flow measurements of the effects of osmotic upshift elicited by glycerol (red line) or KCl (green line) in niosomes composed of unsaturated surfactants plus cholesterol. Buffer (black line) is shown as a control. Representative traces of one out of two independent experiments are shown.</p

Melittin and alamethicin induce calcein leakage.

<p>A: To vesicles filled with 20 mM Na-MOPS, 1 mM CoCl₂ and 0.8 mM calcein, 90 mM NaCl, pH 7.5, 1 μM melittin (final concentration) was added at the time of the black arrow. To obtain the maximum signal, 0.25% (v/v) Triton X-100 was added after 350 s (indicated by the blue arrow). Green lines: niosomes composed of unsaturated surfactants plus cholesterol; red lines: niosomes composed of saturated surfactants plus cholesterol; black lines: liposomes composed of unsaturated lipids plus cholesterol; blue lines: liposomes composed of saturated lipids plus cholesterol. Representative traces of one out of two independent experiments are shown. B: To vesicles as in A, 5 μM alamethicin (final concentration) was added at the time of the black arrow. To obtain the maximum signal, 0.25% Triton X-100 was added after 350 s (indicated by the blue arrow); line color as indicated under A. Representative traces of one out of two independent experiments are shown.</p

Examples of non-ionic surfactants used in this study.

<p>Examples of non-ionic surfactants used in this study.</p

Membrane stability and packing depend on lipid and surfactant composition.

<p>A: Graphical representation of calcein leakage across membrane. B: Leakage of calcein from liposomes and niosomes. Green lines: niosomes composed of unsaturated surfactants plus cholesterol; red lines: niosomes composed of saturated surfactants plus cholesterol; black lines: liposomes composed of unsaturated lipids plus cholesterol; blue lines: liposomes composed of saturated lipids plus cholesterol. Representative traces of one out of two experiment are shown. C: Stability of niosomes and liposomes in the presence of Triton X-100. The data are corrected for dilution; line/symbol color as in B. The upper x-axis is for the liposomes composed of saturated lipids plus cholesterol. Representative traces of one out of two experiment are shown. D: Membrane packing of niosomes and liposomes, measured using the environment-sensitive dye laurdan; line/symbol color as in B. The average of two independent experiments is shown. The spectra of the corresponding vesicles without laurdan were used to correct for background signal.</p

Demographic information of participants.

<p>Demographic information of participants.</p

Correlations among age, weight, BMI, IL-6, IL-8, TNF-α and TGF-β within group 3 (CAD patients without hypertension).

<p>Correlations among age, weight, BMI, IL-6, IL-8, TNF-α and TGF-β within group 3 (CAD patients without hypertension).</p