Kinetic evaluation of free malondialdehyde and enzyme leakage as indices of iron damage in rat hepatocyte cultures. Involvement of free radicals.
The present study relates to the effect of ferric iron supplementation on lipid peroxidation of adult rat hepatocyte pure cultures. Lipid peroxidation was evaluated by free malondialdehyde (MDA) using size exclusion chromatography (HPLC) as a specific and sensitive method. The ferric iron used under its complexed form with nitrilotriacetic acid (NTA) exhibited a prooxidant activity corresponding to an increase of free MDA recovery in the cells and in the culture medium. This enhancement of lipid peroxidation in the hepatocyte cultures supplemented with ferric iron was correlated with an intracellular enzyme leakage (lactate dehydrogenase and transaminase), suggesting that lipid peroxidation and enzyme release represented good parameters for cytotoxicity evaluation. The toxic effect of Fe-NTA on hepatocyte cultures was a function of the incubation time (from 0 to 48 hr) and of the concentration of ferric iron loading (i.e. 5, 20 and 100 microM). The mechanism by which Fe-NTA induced cellular damage involved free radical production, as increasing amounts of free radical scavengers corresponded to diminishing rates of both total free MDA and enzyme release. However, this reducing capacity varied from one scavenger to another, where they exhibited preferentially a decrease in lipid peroxidation or in enzyme leakage. This suggested a dissociation between the two parameters of cytotoxicity considered. Lipid peroxidation corresponding to alterations of both inner membranes and the plasma membrane, whereas enzyme release mainly corresponded to the damage of plasma membrane. Subsequently, some scavengers (superoxide dismutase, mannitol, alpha tocopherol, beta carotene) presented an intracellular activity, as they reduced mostly lipid peroxidation. Other ones (catalase, dimethylpyrroline N-oxide, thiourea) seemed essentially efficient in protecting the external plasma membrane, as shown an important decrease in enzyme leakage.
Morel
I
I
Laboratory of Botany and Cellular Biology, UER du médicament, Rennes, France.
Lescoat
G
G
Cillard
J
J
Pasdeloup
N
N
Brissot
P
P
Cillard
P
P
D000085:Acetates / Q000494:pharmacology*; D000818:Animals; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media; D004705:Endocytosis; D005290:Ferric Compounds / Q000378:metabolism / Q000494:pharmacology*; D005609:Free Radicals; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects*; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008314:Malonates / Q000378:metabolism*; D008315:Malondialdehyde / Q000378:metabolism*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013482:Superoxide Dismutase / Q000378:metabolism
D000085:Acetates; D003470:Culture Media; D005290:Ferric Compounds; D005609:Free Radicals; D008314:Malonates; D008315:Malondialdehyde; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D013482:Superoxide Dismutase; D009571:Nitrilotriacetic Acid; C020326:ferric nitrilotriacetate