Relatively small increases in the steady-state levels of nucleobase deamination products in DNA from human TK6 cells exposed to toxic levels of nitric oxide

Abstract

Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 microM NO along with 186 microM O2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method, and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2'-deoxyuridine (dU) and 2'-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 microM NO and 186 microM O2 for 12 h (1260 microM x min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 h recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI, and dU, respectively, to reach the following levels: dX, 7 (+/- 1) per 10(6) nt; dI, 25 (+/- 2.1) per 10(6) nt; and dU, 40 (+/- 3.8) per 10(6) nt. dO was not detected above the limit of detection of 6 lesions per 10(7) nt in 50 microg of DNA. A 12 h exposure to 0.65 microM NO and 190 microM O2 (468 microM x min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a approximately 15% (+/- 3.6) reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA.

Scheme 1

Spectrum of Products of Nucleobase N-Nitrosation Based on Exposure of Isolated DNA to Nitric Oxide

Scheme 2

Approach to assessing adventitious nucleobase deamination in cellular DNA.

Figure 1

TK6 cell viability (A) and apoptosis (B) as a function of NO dose. Cells were exposed to a steady-state concentration of 1.75 μM NO and 186 μM O₂ for varying periods of time and cell viability and apoptosis measured as described in Materials and Methods. Closed circles: toxicity immediately after exposure; open circles: 24 hr post-exposure. These data represent the mean ± SD for 3-5 measurements.

Figure 2

Tetrahydrouridine (THU) inhibition of dC deaminase activity present in acid phosphatase preparations. Dashed line: dC (1 μg) incubated with acid phosphatase (1.5 units); solid line: dC (1 μg) incubated with acidic phosphatase (1.5 unit) in the presence of THU (3 ng).

Figure 3

Levels of dU (A) and dI (B) in plasmid pUC19 DNA after incubation with TK6 cell extract. (1) Untreated plasmid; (2) plasmid treated with TK6 cell extract; (3) plasmid treated with boiled TK6 cell extract. Data represent mean ± SD of three independent experiments.

Figure 4

Nucleobase deamination in genomic DNA from TK6 cells exposed to steady-state levels of 1.75 μM NO and 186 μM O₂.

Figure 5

The relationship between cytotoxicity and DNA deamination in TK6 cells exposed to various total doses of NO delivered at a steady-state concentration of 1.75 μM. This figure represents an overlay of data from Figures 1 (percent survival, ■) and 4 (dX, ◆; dI, ▼; dU, ●).