Endocytosis-independent uptake of liposome-encapsulated superoxide dismutase prevents the killing of cultured hepatocytes by tert-butyl hydroperoxide.
Liposome-encapsulated (LSOD) or free (FSOD), human recombinant Cu-Zn superoxide dismutase prevented the killing of cultured rat hepatocytes by tert-butyl hydroperoxide (TBHP). A dose of 32 U/ml of LSOD reduced the cell killing by 50%. By contrast, it required 288 U/ml of FSOD to similarly reduce the toxicity of TBHP by 50%. Both LSOD and FSOD increased the cell-associated superoxide dismutase activity of the cultured hepatocytes. Whereas 64 U/ml of LSOD increased cell-associated superoxide dismutase activity fourfold, it required 500 U/ml of FSOD to achieve a similar increase. Furthermore, methylamine, benzyl alcohol, cytochalasin B, oligomycin, and monensin, all inhibitors of endocytosis, prevented the increase in cell-associated superoxide dismutase produced by 500 U/ml of FSOD. These same inhibitors had no effect on the increase in cell-associated superoxide dismutase activity produced by a much lower concentration of LSOD. Thus, liposome-encapsulated superoxide dismutase prevented the cell killing by TBHP more efficiently than free superoxide dismutase because it more efficiently entered the hepatocytes by a mechanism that was independent of the endocytosis responsible for the uptake of FSOD. These data further define the conditions of the toxicity of TBHP. The target hepatocyte must contribute superoxide anions, in addition to the previously shown ferric iron. It is hypothesized that superoxide anions reduce ferric to ferrous iron; the latter then reacts with the hydroperoxide to form tert-butyl alkoxyl radicals. Such radicals are potent oxidizing agents that can initiate the peroxidation of cellular lipids previously shown to lethally injure the hepatocytes.
Nakae
D
D
Department of Oncological Pathology, Nara Medical University, Japan.
Yoshiji
H
H
Amanuma
T
T
Kinugasa
T
T
Farber
J L
JL
Konishi
Y
Y
D000818:Animals; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D066298:In Vitro Techniques; D008081:Liposomes; D008099:Liver / Q000187:drug effects*; D008744:Methylamines / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D010545:Peroxides / Q000037:antagonists & inhibitors*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013482:Superoxide Dismutase / Q000008:administration & dosage*; D020122:tert-Butylhydroperoxide
D001592:Benzyl Alcohols; D008081:Liposomes; D008744:Methylamines; D009840:Oligomycins; D010545:Peroxides; D003571:Cytochalasin B; D008985:Monensin; D020122:tert-Butylhydroperoxide; C027451:methylamine; D013482:Superoxide Dismutase; D019905:Benzyl Alcohol