Copper Deprivation Potentiates Oxidative Stress in HL‐60 Cell Mitochondria

Abstract

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.

Copper, oxidative stress, and human health

“…A Cu deficiency-induced decrease in the activity of CuZn-SOD in humans and animals is well characterized Olin et al, 1994;Strain, 1994;Turnlund et al, 1997;Uriu-Adams et al, 2005), however, protein levels of CuZn-SOD may (Prohaska and Brokate, 2001;West and Prohaska, 2004), or may not (Chung et al, 1988), be reduced. Increased Mn-SOD, which is a well-characterized response to oxidative stress, has been noted in Cu deficient HL-60 cells (Johnson and Thomas, 1999) and Cu deficient animal tissues (Lai et al, 1996).…”

“…Cu deficiency has been shown to decrease SOD activity and increase superoxide anions in Cu deficient rat embryos (Hawk et al, 2003) as well as in adult Cu deficient rats (Lynch et al, 1997). In addition to decreased CuZn-SOD activity, it is postulated that the Cu deficiency-induced decreases in cytochrome c oxidase activity, and the oxidative inactivation of complex I (NADH:ubiquinone oxidoreductase) contribute to the increased production of ROS in Cu deficient animals (Johnson and Thomas, 1999). When rat cardiac myocytes were treated with the Cu chelator diethyldithiocarbamic acid, intracellular superoxide concentrations increased, and apoptosis was triggered suggesting a possible role for oxidative stress in myocardial remodeling (Siwik et al, 1999).…”

“…Mitochondria from Cu deficient neutrophil-like HL-60 cells have increased protein carbonyl groups compared to Cu adequate cells (Johnson and Thomas, 1999). Similarly, Cu deficient mice exhibit increased protein carbonyls in erythrocyte membranes (Sukalski et al, 1997).…”

Association between copper deficiency and DNA damage in cattle

“…Lower Cu/Zn SOD activity with an increase in superoxide anions, lipoperoxides and oxidation of proteins was reported in rats, mice and chickens (Prohaska, 1990;Sukalsky et al, 1997;Cockel and Belonje, 2002;Hawk et al, 2003). Decreased Cu/Zn SOD and cytochrome c oxidase (CCO) activity and increased carbonylated proteins were reported in HL60 cells deprived of copper (Percival, 1998;Johnson and Thomas, 1999). Cattle hypocuprosis was associated with a decrease in Cu/Zn SOD, Cp and CCO activity and with an increase in lipid peroxidation (Xin et al, 1991;Arthington et al, 1996;Ward and Spears, 1997;Gengelbach and Spears, 1998;Cerone et al, 2000a,b).…”

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol–cytochrome c reductase

“…For example, the activity of complex III from rat heart mitochondria has been shown to be reduced in activity in rat hearts due to both aging and ischemia‐reperfusion injury 6, 7. Similarly, other complexes have been identified with compromised function as a function of a variety of physiological states 8, 9. These reductions in activity are routinely attributed to modification of the constituent electron transport complex proteins.…”