Hydrogen peroxide suppresses low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by porcine luteal cells

Abstract

The present study tested the hypothesis that hydrogen peroxide (H 2O 2) inhibits low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by luteal cells. LDL uptake: dispersed porcine luteal cells from mid-cycle (days 6–11, estrus = day 0) were incubated for 0–120 min at 37°C in F-10 medium + 0.1% BSA containing various concentrations of H 2O 2 (0–1000 μM). Cells were washed with catalase (2800 U/ml), and then with fresh medium. Cell viability based on trypan blue exclusion was unaltered by H 2O 2 exposure through 60 min. H 2O 2-exposed cells were incubated with fluorescent-tagged-LDL (Dil-LDL; 1 μg/ml) for 10 min at 37°C. Fluorescence of small (SLC) and large (LLC) luteal cells was analyzed by flow cytometry ( n = 6 experiments). H 2O 2 (≥ 10 μM) caused a progressive reduction ( P < 0.01) in mean fluorescence intensity (MFI) of SLC and LLC indicative of up to a 30–35% decline in LDL uptake. Progesterone (P) production: dispersed luteal cells ( 4 × 10 4 0.2 , ml ) were pre-cultured in DMEM F -2 medium overnight (∼ 18 h) in 96-well culture plates. Wells were rinsed and fresh media (0.2 ml) containing H 2O 2 (0–500 μM) was added. After 30 min, the following treatments were added: human(h)LDL (0 or 50 μg/ml), human chorionic gonadotropin (hCG; 0 or 100 ng/ml), hCG + LDL, or 22 R-hydroxycholesterol (22[OH]-C; 0 or 25 μg/ml), Cells were incubated for an additional 4 h, and P concentrations in final media samples were measured by RIA. P production was increased ( P < 0.05) above basal levels by hCG (1.7×), LDL (2.2×), hCG + LDL (2.9×), and 22(OH)-C (2.4×). H 2O 2 (10–500 μM) dose-dependently suppressed ( P < 0.05) P production in all treatment groups. The relative sensitivities of treatments to H 2O 2 inhibition on the basis of calculated ED 50 (effective doses yielding 50% inhibition) differed as follows: hCG (ED 50 = 72 μM) ≈ LDL (ED 50 = 80 μM) ≈ hCG + LDL (ED 50 = 110 μM) > basal (ED 50 > 500 μM) > 22(OH)-C (ED 50 > 5000 μM). LDL endocytosis/metabolism may be an important target of oxygen radical attack during functional luteal regression.