Effect of Anticancer Quinones on Reactive Oxygen Production by Adult Rat Heart Myocytes

This study investigated the effect of anthracycline antibiotics, mitomycin C, and menadione on oxygen consumption and hydrogen peroxide production by intact, beating, rat heart myocytes. Doxorubicin produced a dose-dependent increase in the rate of cyanide-resistant respiration by beating myocytes. The anthracycline analogs 4-demethoxydaunorubicin, 4 ′ -epidoxorubicin, 4 ′ -deoxydoxorubicin, and menogaril, as well as the anticancer quinones mitomycin C and menadione, also significantly increased oxygen consumption by cardiac myocytes. However, 5-iminodaunorubicin (which has a substituted quinone group) and mitoxantrone (which is not easily reduced by flavin dehydrogenases) had no effect on cardiac respiration. Both catalase (43%) and acetylated cytochrome c (19%) significantly decreased oxygen consumption that had been stimulated by doxorubicin; furthermore, extracellular hydrogen peroxide production was increased from undetectable control levels to 1.30 ± 0.02 nmol/min/107 myocytes ( n = 4 , P < 0.01 ) in the presence of 400 μM doxorubicin. These experiments suggest that the anthracycline antibiotics and other anticancer quinones stimulate cardiac oxygen radical production in intact heart myocytes; such a free radical cascade could contribute to the cardiac toxicity of these drugs.

Structural differences in two biochemically defined populations of cardiac mitochondria

To determine whether there are structural differences in two topologically separated, biochemically defined mitochondrial populations in rat heart myocytes, the interior of these organelles was examined by high-resolution scanning electron microscopy. On the basis of a count of 159 in situ subsarcolemmal mitochondria (SSM, i.e., those that directly abut the sarcolemma), these organelles possess mainly lamelliform cristae (77%), whereas the cristae in in situ interfibrillar mitochondria (IFM, i.e., those situated between the myofibrils, n = 300) are mainly tubular (55%) or a mixture of tubular and lamelliform (24%). Isolated SSM ( n = 374), similar to their in situ counterparts, have predominantly lamelliform cristae (75%). The proportions of crista types in isolated IFM ( n = 337) have been altered, with only 20% of these organelles retaining exclusively tubular cristae, whereas 58% are mixed; of the latter, lamelliform cristae predominate. This finding suggests that, in contrast to SSM, the cristae in IFM are structurally plastic, changing during isolation. These observations on >1,000 organelles provide the first quantitative morphological evidence for definitive differences between the two populations of cardiac mitochondria.

A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress

We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5,8-dihydroxy-1,4-naph-thoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.