Research Article

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 systemSuperoxide production
nmol cytochrome c reduced/min/mg

Control (3)a
 -MicrosomesN.D. (3)b
 -NADPHN.D. (3)
 -Cytochrome cN.D. (3)
Using NADPH (100 μM)N.D. (3)
Using NADH (1 mM) rather than NADPH (3)
Doxorubicin (135 μM) (6)c
 -MicrosomesN.D. (3)d
 -NADPHN.D. (3)d
 -Cytochrome cN.D. (3)d
Using heat-denatured microsomeseN.D. (3)d
Using heat-denatured SODe (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)fN.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.