Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time

Abstract

DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or during the base excision repair pathways. Here we established a new real-time assay to assess an imbalance of DNA SSB repair by indirectly measuring PARP-1 activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye. While this assay is not a direct method, it does not require DNA extraction or alkaline treatment, both of which could potentially cause an artifactual induction of SSBs. In addition, it takes only 4 h and requires less than a half million cells to perform this measurement. Using this assay, we demonstrated that the dose- and time-dependent depletion of NAD(P)H in XRCC1-deficient CHO cells exposed to methyl methanesulfonate. This decrease was almost completely blocked by a PARP inhibitor. Furthermore, methyl methanesulfonate reduced NAD(P)H in PARP-1+/+ cells, whereas PARP-1-/- cells were more resistant to the decrease in NAD(P)H. These results indicate that the analysis of intracellular NAD(P)H level using water-soluble tetrazolium salt can assess an imbalance of SSB repair in living cells in real time.

Figure 1

Intracellular NAD(P)H and NAD⁺ levels in EM9 cells expressing empty vector (EM9-V) or human wild-type XRCC1 (EM9-XH). (A) EM9-XH and EM9-V cells were exposed to MMS for 4 h. Intracellular NAD(P)H levels in living cells were determined by adding CCK-8 solution. (B) EM9-V cells were exposed to MMS for up to 5 h. Intracellular NAD(P)H levels were monitored at 30, 60, 120, 150, 210, 240 and 300 min in real time. (C) EM9-XH and EM9-V cells were exposed to 2 mM MMS for 4 h. Intracellular NAD⁺ levels were determined by the enzyme cycling assay. Mean data and S.D. (bars) were from triplicate experiments using triplicate samples.

Figure 1

Intracellular NAD(P)H and NAD⁺ levels in EM9 cells expressing empty vector (EM9-V) or human wild-type XRCC1 (EM9-XH). (A) EM9-XH and EM9-V cells were exposed to MMS for 4 h. Intracellular NAD(P)H levels in living cells were determined by adding CCK-8 solution. (B) EM9-V cells were exposed to MMS for up to 5 h. Intracellular NAD(P)H levels were monitored at 30, 60, 120, 150, 210, 240 and 300 min in real time. (C) EM9-XH and EM9-V cells were exposed to 2 mM MMS for 4 h. Intracellular NAD⁺ levels were determined by the enzyme cycling assay. Mean data and S.D. (bars) were from triplicate experiments using triplicate samples.

Figure 2

Dose-dependency of the depletion of NAD(P)H in EM9-V and EM9-XH exposed to MMS for 4 h in the absence or presence of PARP inhibitor, 3-AB (10 mM) (A) or DPQ (90 µM) (B).

Figure 3

Depletion of intracellular NAD(P)H in PARP-1^+/+ and PARP-1^–/– fibroblast cells. (A) NAD(P)H level in PARP-1^+/+ and PARP-1^–/– fibroblast cells exposed to MMS for 4 h. Depletion of NAD(P)H in PARP-1^+/+ (B) and PARP-1^–/– (C) fibroblast cells exposed to MMS for 4 h in the absence or presence of 3-AB (10 mM).

Figure 3

Depletion of intracellular NAD(P)H in PARP-1^+/+ and PARP-1^–/– fibroblast cells. (A) NAD(P)H level in PARP-1^+/+ and PARP-1^–/– fibroblast cells exposed to MMS for 4 h. Depletion of NAD(P)H in PARP-1^+/+ (B) and PARP-1^–/– (C) fibroblast cells exposed to MMS for 4 h in the absence or presence of 3-AB (10 mM).

Figure 4

Comparison of the cellular effects induced by MMS detected by NAD(P)H assay and comet assay. Depletion of NAD(P)H in EM9-V cells exposed to MMS for 1 h (A), 2 h (B) and 4 h (C). (D) Tail moments were quantified by the comet assay in EM9-V cells immediately after MMS treatment at 0, 0.0059, 0.0117 or 0.0469 mM for 1 h. Results and S.D. are the mean from triplicate samples.

Figure 4

Comparison of the cellular effects induced by MMS detected by NAD(P)H assay and comet assay. Depletion of NAD(P)H in EM9-V cells exposed to MMS for 1 h (A), 2 h (B) and 4 h (C). (D) Tail moments were quantified by the comet assay in EM9-V cells immediately after MMS treatment at 0, 0.0059, 0.0117 or 0.0469 mM for 1 h. Results and S.D. are the mean from triplicate samples.