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Ascorbic acid at pharmacologically attainable concentrations effectively inhibited the growth of the catecholamine-positive neuroblastoma cell line SK-N-SH; it inhibited LS cells to a smaller extent and catecholamine-negative SK-N-LO cell growth least effectively. In all three cell lines high concentrations of H2O2 were found. Since ascorbic acid was shown to release iron from ferritin in vitro and to keep it in the reduced state, we suggested that it acted as a pro-oxidant in ferritin-rich neuroblastoma cells in the presence of H2O2 and Fe2+ (Fenton reaction), implying iron release from cellular ferritin. We show here that iron could be mobilized from cellular ferritin by 1 mM ascorbic acid in iron-59-preloaded SK-N-SH and LS cells, but not in SK-N-LO cells. In agreement with these results, DNA strand break formation by ascorbate was only observed in SK-N-SH and LS cells. In SK-N-LO cells, DNA strand breaks could be induced by a combination of 1 mM ascorbic acid and 100 microM H2O2. Since cell-damaging effects caused by chemotherapy further facilitate iron release from ferritin, we conclude that ascorbate could be a powerful enhancer of some cytostatic drugs in neuroblastoma therapy.

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A sensitive peroxidase-dependent luminol-enhanced chemiluminescence (ECL) assay for determination of hydrogen peroxide (H2O2) generation by tumour cells was established. This test system allows determination of H2O2 in concentrations as low as 25 pmol (50 nmol/L) and yields results which are comparable to those obtained using a less sensitive photometric method and a previously described scopoletin fluorescence assay. After 3 h incubation time 10(4) SK-N-SH neuroblastoma cells released 60 +/- 5 pmol H2O2 in the supernatant and this level was significantly (p < 0.025) increased by about 70% in the presence of 5 pmol (100 ng/mL) recombinant tumour necrosis factor alpha (TNF alpha). In contrast, H2O2 production was slightly reduced by TNF alpha at a very low concentration of 0.5 fmol (0.01 ng/mL).

The effects of homoeopathic high dilutions of cytokine TNFα on H2O2 production of SK-N-SH neuroblastoma cells were studied. H2O2 was measured using a highly sensitive chemiluminescence method (ECL) based on chemical enhancement of luminol oxidation in the presence of peroxidase and H2O2. The dose-effect relationship between TNFα and H2O2 production of SK-N-SH cells was established over the whole range from 200 ng/ml of TNFα to 100x. Interpolation yielded a waveshaped curve (phase length 5–8) which appeared to ‘ride’ on a wave-shaped 10th order regression curve (phase length ∼ 35). The effects of TNFα 100x on H2O2 production were highly significant (p < 0.002). Elevation of H2O2 production compared to controls was repeatedly seen following incubation of SK-N-SH cells in the presence of TNFα 100x. These results suggest that the chemiluminescence method used is suitable to demonstrate and investigate further the influence of homoeopathic high potencies on biological systems.