Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3030-3034, April 1992 Biochemistry DNA damage and mutation in human cells exposed to nitric oxide in vitro T. NGUYEN*, D. BRUNSON*, C. L. CRESPIt, B. W. PENMANt, J. S. WISHNOK*, AND S. R. TANNENBAUM*t *Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 56-311, Cambridge, MA 02139; and tGentest Corp., 6 Henshaw Street, Woburn, MA 01801 Communicated by Gerald N. Wogan, December 3, 1991 ABSTRACT Nitric oxide (NO') is a physiological messen- EXPERIMENTAL PROCEDURES ger formed by several cell types. Reaction with 02 forms oxides that nitrosate amines at pH values near 7. We now report Materials. Xanthine, hypoxanthine, adenine, guanine, calf thymus DNA, yeast RNA, and transfer RNA (from bovine experiments in which NO' was added to intact human cells and liver) were purchased from Sigma. Heat-denatured DNA was to aerobic solutions of DNA, RNA, guanine, or adenine. TK6 prepared by boiling calf thumus DNA solution (2.0 mg/ml) human lymphoblastoid cells were mutated 15- to 18-fold above for 15 min and quickly chilling it in an ice bath. [1,3- background levels at both the HPRT and TK gene loci. Xan- 15N21Xanthine was purchased from Cambridge Isotope Lab- thine and hypoxanthine, from deamination of guanine and oratories (Cambridge, MA), and N-methyl-N-(trimethylsil- adenine, respectively, were formed in all cases. NO' induced yl)trifluoroacetamide was purchased from Pierce. Nitric ox- dose-responsive DNA strand breakage. Yields of xanthine ide (NO-) was supplied by Matheson or Aldrich. HPLC grade ranged from nearly equal to about 80-fold higher than those of ammonium acetate and organic solvents were from Fisher hypoxanthine. Yields of xanthine and hypoxanthine from nu- Scientific. [14C]Thymidine (57 mCi/mmol; 1 Ci = 37 GBq) cleic acids were higher than those from free guanine and was purchased from ICN. adenine. This was most pronounced for xanthine; 0.3 nmol/mg Mutagenicity Assays. Mutagenicity assays were conducted was formed from free guanine vs. 550 nmol/mg from calf in TK6 human lymphoblasts (10) as described (11). Treatment thymus RNA. Nitric oxide added to TK6 cells produced a 40- with nitric oxide was as follows: 100-ml cultures containing to 50-fold increase in hypoxanthine and xanthine in cellular 4 x 105 cells per ml ofRPMI 1640 medium supplemented with DNA. We believe that these results, plus the expected deami- 10%o horse serum were contained in 500-ml glass Erlenmeyer nations of cytosine to uracil and 5-methylcytosine to thymine, flasks. The mouths of the flasks were covered with sterile account for the mutagenicity of nitric oxide toward bacteria Parafilm. Nitric oxide was introduced directly into the me- and mammalian cells. dium with a sterile stainless steel cannula. The nitric oxide volume was controlled by delivery with a syringe that had Many types of cells produce NO' through a common bio- been purged with nitrogen to remove oxygen. Nitric oxide chemical pathway-i.e., the oxidation of arginine (1, 2). was introduced into the culture at =1 ml/sec, and the vessel Although the function of this NO' is related to its role as a was capped and shaken. Cultures were incubated for 1 h at 370C and then centrifuged at 1000 x g for 5 min. The cell second messenger (3), or perhaps to targeted cytotoxicity (4), pellets were resuspended in 100 ml of the medium and there may also be collateral reactions leading to DNA damage cultured for 6 days to allow phenotypic expression. The in neighboring cells (5, 6). expressed cultures were plated in the presence of trifluo- For many years it has been generally recognized that nitrite rothymidine (three 96-well plates at 30,000 cells per well), in (NO-) can deaminate purines, pyrimidines, and various the presence of 6-thioguanine (three 96-well plates at 30,000 forms of RNA and DNA under acidic conditions; this deam- cells per well), or in the absence of selective conditions (two ination leads to mutations in prokaryotic organisms (7). 96-well plates at 2 cells per well). After a 12-day incubation, These reactions proceed through a pathway in which pro- plates were scored for the presence of a colony in each well. tonation of NO2 leads to formation of N203, an electrophilic Nitric oxide mutagenicity was examined in two independent nitrosating agent. experiments with a total of six replicate cultures for each We proposed (8) that generation of NO' under physiolog- treatment. The positive control was 140 ng of 4-nitroquino- ical conditions in the presence of 02 can reproduce the line-N-oxide per ml. The results of the two independent chemical and mutagenic effects of NOj under acidic condi- experiments were pooled and analyzed according to standard tions. Once formed, N203 (or N204) can react with unpro- methods (11). tonated amino groups in the presence of water (9). Low DNA Strand Breaks. DNA strand breaks in the TK6 cells concentrations of NO- in the presence of 02 in aqueous were detected through a modified version of the DNA solutions may also lead to nitrosyl donors by other mecha- precipitation assay described by Olive (12). Cells in RPMI nisms. 1640 medium supplemented with 10% horse serum were We now demonstrate that, in the presence of 02, NO- can radiolabeled for 20 h with 0.02 uCi of ['4C]thymidine per ml cause a of of to DNA as in 5% CO2 at 37C followed by incubation for 2 h in fresh variety types damage well as serum-free medium. Four milliliters of the radiolabeled TK6 mutations in a human lymphoblastoid cell line in culture. On cells (5 x 105 cells per ml) was placed in a 6-ml vial. the basis of these results, we propose that cytotoxicity and Dose-response experiments were done by introducing vari- mutagenicity associated with the inflammatory process may ous concentrations (0.125, 0.25, and 0.5 ml/ml)§ of nitric result from nitrogen oxide radicals in addition to the better- oxide through the rubber septum into the medium. The known reactions of (or with) oxygen radicals. Abbreviation: TMS, trimethylsilyl. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §At STP 0.1 ml ofNO' equals 2.2 jmol; this was checked by analysis in accordance with 18 U.S.C. §1734 solely to indicate this fact. of nitrate and nitrite in the culture medium. 3030 Downloaded by guest on September 27, 2021 Biochemistry: Nguyen et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3031 sample was mixed and incubated at 370C for 1, 4, and 24 h. The derivatized standards and samples were analyzed on a After incubation, cells (100 ,ul) were lysed in plastic tubes at Hewlett-Packard model 5987 gas chromatograph mass spec- 0C for 1 min in 0.5 ml of solution containing 2% SDS, 10 mM trometer operated in the electron ionization mode. The EDTA, and 10 mM Tris to which 1 ml of 0.05 M NaOH was electron energy was 70 eV. The source temperature was added just prior to lysis. KCI (0.5 ml, 0.12 M) was gently 210'C. Dwell times were 200 msec per ion. The columns were added, and the tubes were capped and placed in a water bath fused-silica capillary, either a 12 m x 0.2 mm HP-1 (Hewlett- at 650C for 10 min, cooled on ice for 5 min, and centrifuged Packard) or a 15 m x 0.25 mm DB-5 (J & W Scientific, for 10 min at 3500 x g. The supernatant was decanted into a Rancho Cordova, CA). The injection port and transfer lines liquid scintillation vial containing 1 ml of 0.05 M HCL. The were at 240'C. The carrier gas was ultra-high-purity helium pellet was resuspended twice in 1 ml of650C water, vortexed, (Medtech, Medford, MA), with a flow rate ofabout 2 ml/min. and poured into a scintillation vial for determination of Samples were injected in the splitless mode with an initial radioactivity by liquid scintillation counting. The percentage oven temperature of 150'C for 2 min, followed by a temper- of DNA damage was expressed as a ratio of radioactivity in ature program to 220'C at 10'C/min, then to 280'C at 250C/ the supernatant to the sum of the radioactivities of the min, with a final hold at 280'C for 2 min. The electron supernatant and the pellet multiplied by 100. Our historical ionization mass spectra are characterized by a base peak at background for DNA single-strand breaks by this method is M - 15. When analyzed at similar concentrations, the nucleic 13.8% ± 4.9% (range = 3.2-24.9%; 95% confidence interval acid bases and the compounds under study were well re- = 12.6-15.0%; n = 67). Experiments in which the back- solved on a 12-m HP-1 fused-silica capillary column. The ground level exceeded 25% were rejected. A statistically levels of the compounds of interest in the DNA hydrolysate different result from background due to treatment requires were small compared to the levels of adenine and guanine, so DNA damage to exceed the 95% upper confidence level of it is necessary to perform HPLC clean-up prior to GC-MS both the historical data and the concurrent control (P < 0.05). analysis. Adenine and guanine can be quantitated simulta- In Vitro neously in the same HPLC separation. Modification of Nucleic Acids and Bases.