Decreased erythrocyte CCS content is a biomarker of copper overload in rats

Abstract

Copper (Cu) is an essential trace metal that is toxic in excess. It is therefore important to be able to accurately assess Cu deficiency or overload. Cu chaperone for Cu/Zn superoxide dismutase (CCS) protein expression is elevated in tissues of Cu-deficient animals. Increased CCS content in erythrocytes is particularly sensitive to decreased Cu status. Given the lack of a non-invasive, sensitive and specific biomarker for the assessment of Cu excess, we investigated whether CCS expression in erythrocytes reflects Cu overload. Rats were fed diets containing normal or high levels of Cu for 13 weeks. Diets contained 6.3 +/- 0.6 (Cu-N), 985 +/- 14 (Cu-1000) or 1944 +/- 19 (Cu-2000) mg Cu/kg diet. Rats showed a variable response to the high Cu diets. Some rats showed severe Cu toxicity, while other rats showed no visible signs of toxicity and grew normally. Also, some rats had high levels of Cu in liver, whereas others had liver Cu concentrations within the normal range. Erythrocyte CCS protein expression was 30% lower in Cu-2000 rats compared to Cu-N rats (P < 0.05). Notably, only rats that accumulated high levels of Cu in liver had lower erythrocyte CCS (47% reduction, P < 0.05) compared to rats fed normal levels of Cu. Together, these data indicate that decreased erythrocyte CCS content is associated with Cu overload in rats and should be evaluated further as a potential biomarker for assessing Cu excess in humans.

Keywords: CCS; biomarker; copper overload; erythrocytes; rat.

Figure 1.

Cu concentrations in liver (A), kidney (B) and plasma (C) of rats fed diets with normal or high amounts of Cu. Each solid circle corresponds to 1 rat, n = 12, 12 and 7 for Cu-N, Cu-1000 and Cu-2000, respectively.

Figure 2.

Relative CCS content in erythrocytes of rats fed the Cu-N, Cu-1000 or Cu-2000 diets. The mean of Cu-N rats was arbitrarily set to 1. CCS expression is expressed relative to GAPDH expression. Values are means ± SEM. Bars without a common letter differ, P < 0.05.

Figure 3.

Relative CCS expression in erythrocytes of rats fed diets with normal amounts of Cu (Cu-N) or high levels of Cu displaying high (High Liver Cu) or normal (Normal Liver Cu) liver Cu concentrations. CCS expression in erythrocytes was quantified and expressed relative to GAPDH expression (A). The mean for Cu-N rats was arbitrarily set to 1. Values are means ± SEM, n = 7, 4 and 10 for Cu-N, High Liver Cu and Normal Liver Cu, respectively. Bars without a common letter differ, P < 0.05. Representative Western blot showing erythrocyte CCS expression in rats fed normal Cu (Cu-N) or rats fed high Cu and having high liver Cu concentrations (High Liver Cu) (B). CCS expression was detected with a CCS-specific antibody (top panel). The membrane was stripped and probed with an antibody against GAPDH (bottom panel).