*3.5.* In Vitro *Repair Assay*

As a final check as to whether vitamin C has any effect on DNA repair activity we used an *in vitro* approach, measuring the ability of extracts of vitamin C-preincubated cells to recognize and incise oxidized bases in substrate nucleoids. HeLa cells were treated with 200 μM vitamin C for 6 h. Extracts were prepared and incubated for 10 or 20 min with gel-embedded HeLa nucleoid DNA containing oxidized purines. (Previous experiments [13] have shown no significant breaks induced in undamaged substrate.) Then the comet assay was performed to measure the breaks induced by the extracts, as an indicator of repair activity. Extracts from HeLa cells preincubated with 200 μM vitamin C produced a significant increase in SBs in substrate nucleoids (compared with extracts from non-vitamin C-treated cells) at both reaction incubation times (Figure 6A). Lower concentrations of vitamin C (50 or 100 μM) did not have any effect. However, when we checked the possibility that vitamin C was having a direct effect on the DNA, we found that breaks were in fact induced in HeLa nucleoid DNA containing oxidized purines or in undamaged DNA (Figure 6B,C). A significant effect is seen at a concentration as low as 1 μM. This concentration relates to the concentration as prepared in buffer A, before 5× dilution with buffer F. Therefore the concentration capable of damaging DNA is actually as low as 0.2 μM.

**Figure 6.** (**A**) Effect of extracts from HeLa cells pre-treated with 0 and 200 μM of vitamin C for 6 h on a DNA substrate containing oxidized purines. (**B**) Effect of different concentrations of vitamin C on a DNA substrate containing oxidized purines. (**C**) Effect of different concentrations of vitamin C on a non-damaged DNA substrate. In (**A**), Buffer F was used as a negative control. In (**A**) and (**B**), where nucleoids contained oxidized bases, FPG was employed as a positive control. Concentrations of vitamin C displayed in the box on panels (**B**) and (**C**) refer to concentrations in buffer A: final concentrations were 4× less. Bars: SD calculated from the results of 3 independent experiments. Statistical comparisons (\* *p* < 0.05): vitamin C (different concentrations) *vs*. 0 μM.
