Iron loading of cultured hepatocytes. Effect of iron on 5-aminolaevulinate synthase is independent of lipid peroxidation

Abstract

Cultured chick embryo hepatocytes were iron-loaded with ferric nitrilotriacetate. Iron-loading was confirmed by both quantitative cellular iron determinations and ultrastructural studies. With iron-loading, lipid peroxidation, as detected by malonaldehyde released into the medium, occurred at a linear rate for 12h, after which time the rate of malonaldehyde production decreased. No cell toxicity, as detected by lactate dehydrogenase release, was noted. The amount of malonaldehyde recovered in the medium after 18h of exposure to iron represented 24-33% of the total malonaldehyde that could be produced by incubating lysed cells with iron and ascorbate. Cellular glutathione was not affected by iron-stimulated lipid peroxidation, but was increased by allylisopropylacetamide. Although iron-loading by itself had no effect on activity of 5-aminolaevulinate synthase, the first and rate-limiting step in haem synthesis, iron-loading in the presence of the porphyrogenic drug allylisopropylacetamide increased levels of 5-aminolaevulinate synthase 6-fold over levels induced by the drug alone. The antioxidant, butylated hydroxytoluene, totally inhibited iron-stimulated lipid peroxidation, but did not interfere with the effect of iron-loading to potentiate an increase in 5-aminolaevulinate synthase. After 18h of exposure to iron, followed by a change to fresh medium, the iron remaining within the cells did not stimulate further lipid peroxidation over the following 18h, but did potentiate an increase in 5-aminolaevulinate synthase on exposure to allylisopropylacetamide. It therefore appears that lipid peroxidation is not the mechanism by which iron potentiates induction of hepatic 5-aminolaevulinate synthase.

Iron Stimulates Urokinase Plasminogen Activator Expression and Activates NF-kappa B in Human Prostate Cancer Cells

“…12-tetradecanoylphorbol-13 acetate (phorbol ester, PMA; Sigma-Aldrich; St. Louis, MO) was reconstituted in ethanol at a concentration of 1 mg/ml, aliquotted and stored at -70 • C. 2 ,7 -dichlorofluorescein diacetate (DCFDA; Molecular Probes, Eugene, OR) was prepared as a 5 mM stock solution in DMSO, aliquotted and stored at -70 • C. Anti-human transferrin receptor antibody (Oncogene Research Products, Calbiochem; San Diego, CA) was reconstituted in PBS at a concentration of 100 µg/ml, aliquotted and stored at -70 • C. Ferric nitrilotriacetate (FeNTA) pH 7.4, was prepared from 5 mM ferric citrate and sodium nitrilotriacetate (NTA) as described previously (23), and was kindly provided by Dr. Peter Sinclair. FeNTA and NTA solutions were stored at 4 • C. All solutions that were added to cell cultures were sterile filtered though 0.2 µM filters.…”

ARSENITE DECREASES CYP3A4 AND RXRα IN PRIMARY HUMAN HEPATOCYTES

“…Proteins were determined by the method of Lowry et al (1951) using bovine serum albumin as the standard. Lipid peroxidation in hepatocytes and media was measured by the generation of thiobarbituric acid reactive substances (TBARS), as described (Shedlofsky et al, 1983).…”

“…The final concentration of dimethyl sulfoxide in culture medium was below 1 l/ml. FeNTA was prepared as described (Shedlofsky et al, 1983).…”

“…Effect of rifampicin, sodium arsenite, and ferric nitrilotriacetic acid on lipid peroxidation in primary human hepatocytes. Following 24 h with no treatment (control) or treatment with the chemicals indicated, TBARS were measured in media and cells, as described (Shedlofsky et al, 1983). Values represent the mean Ϯ S.D.…”

High-throughput non-heme iron assay for animal tissues

“…No. M376) and nitrilotriacetic acid (Sigma, St. Louis, MO) as previously described [11]. In the cuvette-based assay, 100 AL of each FeNTA dilution (or blank) was added to 1 ml of color reagent.…”