Mitochondrionopathy Phenotype in Doxorubicin-Treated Wistar Rats Depends on Treatment Protocol and Is Cardiac-Specific

Abstract

Although doxorubicin (DOX) is a very effective antineoplastic agent, its clinical use is limited by a dose-dependent, persistent and cumulative cardiotoxicity, whose mechanism remains to be elucidated. Previous works in animal models have failed to use a multi-organ approach to demonstrate that DOX-associated toxicity is selective to the cardiac tissue. In this context, the present work aims to investigate in vivo DOX cardiac, hepatic and renal toxicity in the same animal model, with special relevance on alterations of mitochondrial bioenergetics. To this end, male Wistar rats were sub-chronically (7 wks, 2 mg/Kg) or acutely (20 mg/Kg) treated with DOX and sacrificed one week or 24 hours after the last injection, respectively. Alterations of mitochondrial bioenergetics showed treatment-dependent differences between tissues. No alterations were observed for cardiac mitochondria in the acute model but decreased ADP-stimulated respiration was detected in the sub-chronic treatment. In the acute treatment model, ADP-stimulated respiration was increased in liver and decreased in kidney mitochondria. Aconitase activity, a marker of oxidative stress, was decreased in renal mitochondria in the acute and in heart in the sub-chronic model. Interestingly, alterations of cardiac mitochondrial bioenergetics co-existed with an absence of echocardiograph, histopathological or ultra-structural alterations. Besides, no plasma markers of cardiac injury were found in any of the time points studied. The results confirm that alterations of mitochondrial function, which are more evident in the heart, are an early marker of DOX-induced toxicity, existing even in the absence of cardiac functional alterations.

Cardiac cytochrome c and cardiolipin depletion during anthracycline-induced chronic depression of mitochondrial function

“…The treatment protocol used followed the sub-chronic procedure previously established by Solem et al (Solem et al, 1996) and adapted by us in a different strain of rats (Pereira et al, 2012). Briefly, 8 weeks old (total n = 35) male Wistar rats, Crl:WI(Han), purchased from Charles River (France), were randomly grouped in pairs and received weekly subcutaneous injection in the scruff or flank of either DOX (2 mg/Kg) or an equivalent volume of NaCl 0.9% during seven weeks, and allowed to rest one week after the last injection.…”

“…Mitochondria from the three different tissues (heart, liver and kidney) were isolated by differential centrifugation according to the standard procedure used in our laboratory (Pereira et al, 2012). Mitochondrial protein was quantified by the biuret method (Gornall et al, 1949) using BSA as a standard.…”

“…We have previously characterized a sub-chronic rodent model of DOX-induced toxicity and reported that alterations in mitochondrial physiology occur in the absence of changes in cardiac biochemical markers or cardiac structure/function assessed by echocardiogram (Pereira et al, 2012), representing a sub-clinical marker of DOX cardiotoxicity.…”

“…Both events, of still unknown origin, appear to be cardio-selective. Hence, in the present investigation we intend to use a multi-organ approach (Pereira et al, 2012) to compare and contrast the effects of DOX-treatment in heart versus liver and kidney, in order to ascertain the drug selectivity towards cardiac mitochondria.…”

“…Both hypotheses were tested in a sub-chronic rodent model of DOX treatment previously established by Solem et al (Solem et al, 1996) and currently in use by us (Pereira et al, 2012). This model shows cardiac mitochondrial alterations, which appear before significant ultrastructure alterations.…”