The various faces of copper in laboratory animals

Abstract

All research described in this thesis focuses on the role of copper in various biochemical processes. It appears that copper has various faces in laboratory animals. On the one hand, copper is an essential trace element, which implicates that a certain requirement for copper exists. On the other hand, copper may be involved in the formation of free radicals and reactive oxygen species (ROS), causing the development of oxidative stress. Oxidative stress has been associated with reduced lifespan and various diseases as a consequence of oxidative damage at the (sub)cellular level. Copper may not only affect biochemical processes in laboratory animals, it may also be affected itself by endogenous and exogenous factors, such as dietary cholesterol. Furthermore, strain differences in hepatic copper content and hepatic copper concentration have been found, which can be (partly) explained by genetic differences. All studies described in this thesis, except the review in chapter 3, were performed in laboratory animals keeping the 3 R’s of laboratory animal science (reduction, refinement and replacement) in mind. Some of the studies performed are for the benefit of the animal (chapter 2), in some studies the animal is used as a model (chapter 6-10) and in some studies, the animal was used as a model, but the results of the study could be used for the benefit of the animal (chapter 4 and 5). In case the animal was used as a model, the purpose of the experiment was to study mechanisms in vivo rather than in vitro. The overall conclusions are : (I.) Copper is an essential trace element, implicating that a certain copper requirement exists to compensate for endogenous losses. The dietary copper allowance for the NMRI outbred laboratory mouse, as determined in the study described in chapter 2, is 4 ppm Cu, which is lower than the NRC’s estimated allowance of 6 ppm Cu for maintenance and 8 ppm for growing and lactating mice, but in line with results described by other authors. The difference in dietary copper allowance probably stems from the fact that the recommendation of the NRC is based on rats and on four studies in mice, of which three were not designed to study copper requirement. We feel that this study contributes to a soundly based estimated copper allowance for mice. (II.) Evidence for the involvement of copper in the formation of free radicals and ROS comes mainly from in vitro research (chapter 3). No evidence was found for copper-mediated oxidative damage at the (sub)cellular level (chapter 4) nor a reduced lifespan was found (chapter 5) in mice fed diets with increasing copper concentrations. These results raise serious questions about the likelihood of developing oxidative stress in other rodents or in human in vivo after high copper intakes. (III.) In literature, dietary cholesterol has been associated with reduced liver copper concentrations and/or liver copper content. Feeding a cholesterol-rich diet to rats did not affect the liver copper content. In rabbits, a decrease in the hepatic copper store was found only in the dietary cholesterol-susceptible inbred strain. The idea that cholesterol-susceptible animals will show a greater decrease in hepatic copper content after being fed a cholesterolrich diet thus could not be consistently confirmed. In both rabbits and rats a decrease in liver copper concentration was found, but the decrease in rats was probably due to diet-induced hepatomegaly. The reduced liver copper concentrations in rats fed a cholesterol-rich diet as described in literature may also be the result of dietary-induced hepatomegaly. (IV.) Strain differences in liver copper store and liver copper concentrations can (partly) be explained by genetic differences between the strains. QTL analysis can be helpful in identifying genes that are involved in such quantitative traits. Some of the QTLs found in rats may give a clue as to what genes are involved in copper regulation. In the rabbit, however, more research on the structure of the genome is needed before candidate genes for QTLs can be identified.