Copper-Metallothionein from the Toxic Milk Mutant Mouse Enhances Lipid Peroxidation Initiated by an Organic Hydroperoxide

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The toxic milk mutation is an autosomal recessive mutation found in an inbred C57BL/6J strain of mice which results in an excessive hepatic accumulation of copper (Cu), mostly associated with metallothionein (MT). The possible toxicological significance of elevated levels of hepatic copper-metallothionein (Cu-MT) was assessed. The effects of Cu-MT on lipid peroxidation (LP) initiated by an organic peroxide were investigated in an in vitro rat liver microsomal incubation system. Addition of Cu-MT (3 μM) could significantly enhance (100%) LP induced by addition of tertiary-butyl hydroperoxide (t-BHP, 0.1 mM). Similar incubations of Cu-MT in the absence of t-BHP showed negligible LP. Addition of copper sulfate at high concentrations (100 μM) also increased t-BHP induced LP, but the enhancement was less pronounced than observed with Cu-MT addition. Chelation of copper with bovine serum albumin and triethylenetetramine tetrahydrochloride eliminated the enhancement of LP by Cu-MT. Evidence is provided on degradation of MT and release of free Cu in the incubation system. Additions of deferoxamine were also found to prevent LP. Therefore, chelatable Cu, released from Cu-MT, may be responsible for the enhancement of the iron-dependent LP in this system but Cu-MT alone in the absence of iron cannot initiate LP. These in vitro results suggest that conditions resulting in high cellular levels of Cu-MT may exhibit a predisposition to oxidative stress.