Mechanisms of Anthracycline-Enhanced Reactive Oxygen Metabolism in Tumor Cells

Table 2

Requirements for doxorubicin-stimulated superoxide formation in the tumor microsomal fraction. Superoxide production in tumor microsomes was determined in paired, 1 ml reaction mixtures which contained 150 mM potassium phosphate buffer, pH 7.4, 100 μM EDTA, 56 μM acetylated cytochrome c, 200 μg of microsomal protein, and either 0 or 10 μg of SOD. The chemotherapeutic agent was added to the paired reaction mixtures, where specified, before the initiation of the reaction by addition of NADPH (1 mM).

Experimental system	Superoxide production
	nmol cytochrome c reduced/min/mg
Control	(3)^a
-Microsomes	N.D. (3)^b
-NADPH	N.D. (3)
-Cytochrome c	N.D. (3)
Using NADPH (100 μM)	N.D. (3)
Using NADH (1 mM) rather than NADPH	(3)
Doxorubicin (135 μM)	(6)^c
-Microsomes	N.D. (3)^d
-NADPH	N.D. (3)^d
-Cytochrome c	N.D. (3)^d
Using heat-denatured microsomes^e	N.D. (3)^d
Using heat-denatured SOD^e	(3)
Using NADPH (100 μM)	(6)
Using NADH (1 mM) rather than NADPH	(3)^d
Using NADP^+ (1 mM) rather than NADPH	(3)^d
Using NAD^+ (1 mM) rather than NADPH	(3)^d
Using FAD (1 mM)^f rather than NADPH	(3)^d
Using FMN (1 mM)^f	N.D. (3)^d
+DMSO (13 mM)	(3)
+Catalase (1500 units)	(3)
+Dicumarol (10 μM)	(3)

^a.; number in parentheses is number of experiments performed; ^bN.D. is not detectable; ^csignificantly different from control (); ^dsignificantly different from complete system containing NADPH and doxorubicin (); ^emicrosomes or SOD heated for 60 min in a boiling water bath; samples containing heat-denatured SOD were paired against identical mixtures with native dismutase; ^fFAD: flavin adenine dinucleotide; FMN: flavin mononucleotide.