Mechanisms of Anthracycline-Enhanced Reactive Oxygen Metabolism in Tumor Cells

Table 6

Requirements for anthracycline-enhanced superoxide anion production by the tumor mitochondrial fraction. Superoxide formation in the tumor mitochondrial fraction was examined using paired, 1 ml reaction mixtures containing 250 mM sucrose, 20 mM HEPES, pH 8.2, 100 μM EDTA, 56 μM acetylated cytochrome c 100 μg of mitochondrial protein, and either 0 or 10 μg of SOD. The reaction mixture was preincubated for 5 min at 37° with 4 μM rotenone before initiation of the reaction with 100 μM NADH.

Reaction mixture	Superoxide production (nmol/min/mg)
Control	(7)^a
-NADH	N.D. (3)^b
-Mitochondrial fraction	N.D. (3)
-Rotenone	(3)
Using NADPH (100 μM) rather than NADH
Doxorubicin (135 μM)	(10)^c
-NADH	(3)^d
-Mitochondrial fraction	(3)^d
Using heat-denatured mitochondria	(3)^d
-Rotenone	(3)^d
Using NADPH (100 μM) rather than NADH	(3)^d
-Acetylated cytochrome c	N.D. (3)^d
-EDTA	(3)
Using heat-denatured SOD	(3)
Daunorubicin (135 μM)	(3)^c
Rubidazone (135 μM)	(3)^c
Aclacinomycin A (135 μM)	(3)^c
5-Iminodaunorubicin (135 μM)	(3)

^a; numbers in parentheses are numbers of experiments; ^bN.D. is not detectable; ^csignificantly different from control (); ^dsignificantly different from complete system containing doxorubicin alone ().