Bleomycin assay

Bleomycin, an antitumour antibiotic, binds iron in the reduced, divalent state to its secondary amide group. It binds low molecular weight iron ions (often loosely referred to as free iron) in forms that catalyse free radical reactions (Gutteridge et al., 1981) but not iron bound within native, functional protein structures, such as ferritin, transferrin, lactoferrin, haemoglobin, myoglobin, cytochromes etc. The resulting bleomycin-iron complex is capable of degrading DNA 

The bleomycin assay has been applied to assess the levels of available, nontransferrin-bound iron in plasma samples in patients with iron overload disease (Peters et al., 1985), with acute leukaemia before and after drug chemotherapy (Halliwell et al., 1988), as well as patients with rheumatoid arthritis (Winyard et al., 1987). 

Treatment of DNA with iron chelators such as EDTA, DETAP AC or desferrioxamine protects it from degradation. Thus, the destruction of DNA by bleomycin is iron-dependent. The breakdown of bleomycin may be initiated by a ferryl radical species or a hydroxyl 

Presumably, if the reaction is mediated by the formation of a hydroxyl radical, it is formed so close to the DNA that it cannot be relatively more accessible to the added antioxidant to be scavenged effectively. 

All glassware must be rinsed with iron-free distilled water before use. 

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Fig. 4.2. Procedure for the bleomycin assay for non-haem iron. Reagents must be added strictly in the order of the direction of the arrow.

See Figure 2. BALB/c mice with a tumor in one flank and an abscess in the other were assayed 24 h after injection the data are reported as percent of injected radioactivity per gram of tissue. Data in the third and fourth columns are very different, implying that the EDTA moiety becomes separated from the bleomycin moiety in vivo. It is interesting to note that the third column roughly parallels the second column (they would be identical if the experiments were perfect) and that the fourth column parallels the first. 

Cu(II) bleomycin was also analyzed using the FID assay (Figure 4). In stark contrast to the others, this metalloWeomycin failed to produce an appreciable decrease in overall F and did not exhibit FID plot curvature. Notably also, Cu(II) bleomycin did not discriminate against binding to the A/T-only cassettes the A/T-only cassettes were distributed throughout the oligonucleotide rank order in contrast to the other metallobleomycins studied. These results indicated that Cu(II) bleomycin did not bind to these hairpin substrates appreciably under... 

The preceding FID analysis of several metallobleomycins verified that the DNA binding site-selectivities of Co(III) and Fe(III) bleomycin parallel each other quite closely and also correlate to the established cleavage site-selectivity of Fe(II) bleomycin + O2. Notably different, Cu(II) bleomycin did not exhibit an FID assay response under conditions used for the other metallobleomycins. This outcome was likely due to the weaker DNA binding and fast exchange of Cu(II) bleomycin relative to other metallobleomycins (48) and was no doubt exacerbated by the presence of a competitive binding fluorophore. In spite of this limitation, the FID assay is likely to contribute to the study of additional metal complex-DNA interactions in the future. 

Chicken testis cells at different stages of spermatogenesis, separated by centrifugal elutriation as described in (7), and spermatozoa obtained from the vas deferens, were treated with bleomycin for 1 hr and the poly(ADP-ribose) polymerase activity was assayed in nuclei isolated from the different cell t)rpes. Incubation with bleomycin resulted in stimulation of the enzymatic activity in premeiotic and meiotic cells, round spermatids and in elongated spermatids and was ineffective in spermatozoa (Fig. 1). 

Fig. 3. (left) Effect of bleomycin treatment on NAD content in cells at successive stages of spermatogenesis. Tbe NAD content was assayed in the acid-soluble extracts. I n, premeiotic and meiodc cells m, round spermatids FV, elongated spermatids V, spermatozoa from the vas deferens. Control cells (dotted bars), cells treated with bleomycin for 1 hr (hatched bars). 

Pohl, H., and Reidy, J. A., 1989, Vitamin C intake influences the bleomycin-induced chromosome damage assay Implications for detection of cancer susceptibility and chromosome breakage syndromes, Mutat. Res. 224 247-52. 

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