Detection and measurement by high-performance liquid chromatography of malondialdehyde crosslinks in DNA

ANALYTICAL

BIOCHEMISTRY

Detection

143, 265-271

(1984)

and Measurement by High-Performance of Malondialdehyde Crosslinks F. W. SUMMERPIELD

Department

of Food

Science

and Technology,

Liquid Chromatography in DNA

AND A. L. TAPPEL University

of California,

Davis,

California

95616

Received May 3, 1984 Malondialdehyde, which is generated by lipid peroxidation, can form DNA-protein and/or interstrand DNA crosslinks. The biological consequences of inaccurate repair of these crosslinks may be severe. The expected levels of crosslinking of DNA in vivo are low, and an extremely sensitive method must be used for their detection and measurement. Because both types of crosslinks may contain cytosine, the cytosine residues of DNA were labeled in vitro with 12r1. The iodinated DNA was treated with Penicillium nuclease P, at pH 6 and with alkaline phosphatase at pH 9, and the nucleosidic compounds were analyzed by high-performance liquid chromatography. The optimum conditions for measurement of the crosslinks on Ultrasphere ODS or Zorbax ODS columns were 50 mM ammonium phosphate buffer, pH 7, that contained 2% methanol and 5 mM tetra-t-butylammonium phosphate. Both DNA-protein and interstrand DNA crosslinks were measurable simultaneously. The method was quantitative, reproducible, and able to detect crosslinked adducts at subpicomole levels, so that as few as two crosslinks per 106 base pairs were detectable. o 1984 Academic RW IIIC.

only a few crosslinks per million base pairs and so cannot be detected by fluorescence. Current methods of measuring crosslinks in DNA are not sensitive enough to detect this low level of crosslinking (18), or can determine only relative amounts of crosslinking (19). The method presented in this paper uses radiolabeling of the DNA, enzymatic digestion of the DNA, separation of the labeled crosslinks from the undamaged bases by HPLC, and detection by flow liquid scintillation counting in order to obtain the sensitivity needed for the measurement of these crosslinks at the level at which they are expected to occur in viva.

Damage to DNA can be caused by lipid peroxidation (l), which can be initiated by radiation (2), environmental pollutants (3), oxidant drugs (4), halocarbons (5), or vitamin E deficiency and/or high dietary polyunsaturated fat (6). Malondialdehyde (MDA),’ a major product of lipid pet-oxidation, is more damaging to DNA than are other products of lipid peroxidation because it forms crosslinks on the DNA (1) and may also form DNA-protein crosslinks (7-9). It is carcinogenic ( 10) and has caused mutagenesis ( 11) and sister chromatid exchanges (9) in eukaryotic cells. Other chemical agents (13-l 6) can crosslink DNA, and the repair of these crosslinks is much more difficult than the repair of damage caused by modification of single bases (17). The crosslinks formed by MDA on DNA are fluorescent (1); however, the amount of crosslinking needed to cause serious cellular impairment or death may be

MATERIALS

AND METHODS

Materials. DNA from herring sperm and calf thymus, Penicillium nuclease Pi, alkaline phosphatase, and cytochrome c were purchased from Sigma, St. Louis, Missouri, and proteinase K was from Boehringer-Mannheim, Indianapolis, Indiana. 1,3Propanediol, obtained from Eastman Kodak Company,

’ Abbreviations used: MDA, malondialdehyde; IdC, [‘251]iododeoxycytidine; PIC A, tetra-t-butylammonium phosphate. 265

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$3.00

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Rochester, New York, was distilled twice; only the fraction that distilled at over 200°C was used, Na1251 was obtained from Amersham, Arlington Heights, Illinois. HPLCgrade methanol was purchased from Burdick and Jackson, Muskegon, Michigan, and tetrat-butylammonium phosphate (PIC A) from Waters Associates, Milford, Massachusetts. Crosslinking of DNA. MDA was prepared from 1,3-propanediol as previously described (20) and concentrated to lo-20 mM before it was used. Herring sperm DNA was crosslinked via a modification of a previously described method (21). The reaction was carried out under nitrogen at 50°C in pH 5 buffer (50 mM sodium acetate, 0.5 M NaCl, 1 mM CaC12, and 1 mM sodium azide). The concentration of DNA, given in base pairs, ranged from 2-10 mM in different experiments; the concentration of MDA ranged from l-5 mM; and the total volume of each reaction mixture was l-3 ml. After the reaction had proceeded for 3-5 days, the DNA was purified by chromatography on a 1 X 25cm column of Bio-Gel A- 1.5 and eluted with 10 mM sodium phosphate buffer, pH 7. Absorbance was monitored at 254 nm, and the fluorescence of the fractions was measured at 390 nm excitation and 460 nm emission. The fractions showing fluorescence and, therefore, containing the crosslinked DNA were pooled and concentrated for further use. Calf thymus DNA was similarly crosslinked and purified. Zodination of DNA. Native calf thymus DNA and crosslinked samples of the herring sperm or calf thymus DNA were iodinated according to Orosz and Wetmur’s modification (22) of Commerford’s procedure (23) which is specific for cytosine. The DNA was denatured by heating to 95°C for 5 min in 0.1 M sodium acetate buffer, pH 5, in a lml vial with a septum. The amount of DNA varied from 10 to 500 nmol, and after the solution had cooled to 25-3O”C, an amount of Na’251 50% greater than the cytosine content of the DNA was added in the presence of a sixfold excess of thallic chloride. The

AND

TAPPEL

specific activity of the ‘25I was 104- 1O6 cpm/ nmol. After the reaction mixture was heated for 45 min at 70°C an amount of sodium metabisulfite greater than that calculated to reduce the thallic chloride was added, and the mixture was heated for an additional 30 min. The effects of pH on the digestion of iodinated DNA were determined by incubating aliquots of iodinated DNA with nuclease P, at 37°C for 2 h in 1 ml of 25 mM phosphoric acid, 25 mM sodium acetate, and 25 mM N-ethylmorpholine buffer at pH values from 4 to 9. Trichloroacetic acid was added to lo%, and the precipitate was collected by filtration of the solution through nitrocellulose filters. The effect of buffer concentration on the digestion of the DNA was found similarly, using histidine buffer at pH 6. For the effect of pH on the elution of iodinated DNA from nitrocellulose filters, identical aliquots of the DNA that had been precipitated with ethanol and collected on the filters were eluted with 2 ml of the same buffer that was used to ascertain the optimum pH for digestion by nuclease. The effects of buffer concentration on elution were determined similarly, using histidine buffer at pH 8. The DNA from single iodinated samples was recovered by chromatography on a 1 x 25-cm column of Sepharose 6B eluted with 10 mM sodium phosphate buffer, pH 7. The fractions that contained the labeled DNA were concentrated and digested at 37°C with nuclease Pi for 6-8 h at pH 6, and then with alkaline phosphatase for 12-16 h at pH 9. The digest was chromatographed on a 1 x 25-cm column of Bio-Gel P-2 and eluted with 10 mM sodium phosphate buffer, pH 7. Peaks containing radioactivity ([ 1251]iododeoxycytidine, IdC) and fluorescence (crosslinked adducts) were concentrated for further use. An alternate method was used for processing up to 12 samples of DNA simultaneously. The DNA was recovered by transferring the

CHROMATOGRAPHIC

MEASUREMENT

OF

reaction mixture to a test tube, adding 3 vol of ethanol, and cooling to - 15°C. The precipitate was collected by filtration and, unless otherwise stated, was eluted from the filters by agitation with a vortex mixer in a small test tube with 2 ml of 50 mM histidine buffer, pH 8, that contained 1 mM sodium azide. The samples were digested as described before, and the enzymes were precipitated by adding 3 vol of ethanol and cooling the mixture to -15°C. The filtrate, which contained the nucleosidic compounds, was concentrated by heating with a 250-W infrared lamp in a desiccator with the pressure maintained at 30 Torr; the temperature of the samples reached about 45°C. After the filtrate was concentrated to 0.5 ml or less, it was stored at - 15°C for further use. The IdC and the labeled, putative interstrand DNA crosslink formed by MDA between IdC and deoxyguanosine (IdC-MDAdG) were prepared by chromatographing digests of the iodinated, crosslinked herring sperm DNA. The labeled adduct formed by the crosslinking of cytosine to the E-amino group of lysine by MDA (IdC-MDA-Lys) was prepared by crosslinking the purified IdC to cytochrome c by MDA. The conditions were the same as those used for the crosslinking of the DNA, except that the 0.5 M NaCl was omitted and 2 mM IdC and 0.5 mM cytochrome c were used. The modified, fluorescent cytochrome c was recovered by chromatography on a 1 X 25-cm column of Bio-Gel P- 10 and elution with 10 mM sodium phosphate buffer, pH 7, and was digested at 37°C for 12-16 h with proteinase K in 25 mM Tris, pH 8, that contained 0.5 M NaCl and 5 mM EDTA. The digest was chromatographed on a 1 X 25-cm column of Bio-Gel P-2 and eluted with 10 mM sodium phosphate buffer, pH 7. The fractions that contained a high level of fluorescence and radioactivity were pooled and concentrated for further use. HPLC. Unless otherwise stated, the eluent was 50 mM ammonium phosphate, pH 7. For some experiments, the buffer contained up to 20% methanol and/or 10 mM PIC A.

MALONDIALDEHYDE-DNA

CROSSLINKS

267

Most studies were performed on a 0.46 X 25cm Ultrasphere ODS column at a flow rate of 0.5 ml/min. The absorbance of the eluate was monitored at 254 nm; the volume of the flow cell in the detector was 8 ~1. The fluorescence of the eluate was monitored by passing it through a 50-~1 flow cell in an Aminco-Bowman spectrophotofluorometer. When preparations that contained only interstrand crosslinks were analyzed, the wavelength settings were the same as those used for the measurement of the fluorescence of individual fractions; when the putative DNAprotein crosslink was also expected, the excitation monochromator was set at 375 nm. For some experiments, the eluate was collected in l-ml fractions and the radioactivity in an aliquot of each fraction was counted by liquid scintillation. These data were smoothed for plotting and integration by a computerized curve-fitting procedure. In other experiments, radioactivity was measured by flow liquid scintillation counting. For the studies in which the sensitivity of the method was tested, a 0.46 X 25-cm Zorbax ODS column was used with a flow rate of 0.5 ml/min. The eluate or a portion thereof was mixed with scintillation fluid and pumped through a 2-ml glass coil in the counting chamber of a scintillation spectrometer. The output of the spectrometer was converted to an analog signal by a ratemeter and recorded, and the peaks were integrated for measurement of radioactivity. RESULTS Sample preparation. When the procedure summarized in Fig. 1 was used for more than 20 iodinations of either herring sperm or calf thymus DNA, regardless of the extent of crosslinking, about 70% of the theoretical yield of radioactivity was incorporated into the DNA. Recovery of the DNA from the reaction mixture was greater than 95% when it was purified by chromatography, and only negligible amounts of the radioactivity in the fractions that contained the DNA could be

SUMMERFIELD

268 ISOLATED

I

DNA

LABEL WITH lz5 I, PRECIPITATE WITH 3 VOL. ELUTR FROM FILTERS WITH 50 mH - HISTIDINE, SODIUM AEIDE (pli 8)

LABELED

ETHANOL, 1 mH -

DNA

ADJUST To pH 6, DIGEST WITH NUCLEASE

J.

LABELED, CROSSLINRED NUCLEOTIDES, UBLABELED NUCLEOTIDES

IdC

P 1 5'-PHOSPHATE,

ADJUST TO pH 9, DIGEST WITH ALKALINE LABELED, CROSSLINKED NUCLEOSIDES

NUCLEOSIDES,

IdC,

PHOSPHATASE UNLABELED

PRECIPITATE WITH 3 VOL. ETHANOL, FILTER CONCENTRATE BY EVAPORATION AT 30 T0P.R CONCENTRATED

SAMPLE FOR ANALYSIS

AND

BY HPLC

FIG. 1. Preparation of radiodinated nucleoside sample for HPLC.

removed by dialysis. When the DNA was recovered by precipitation, filtration, and elution from the filters with 50 mM histidine, 1 tnM sodium azide, pH 8, 95% of the nondialyzable radioactivity that was precipitated was eluted from the filters. Digestion of the DNA and subsequent dephosphorylation of the nucleotides were over 90% complete. Over 90% of the sum of 5-iodocytosine and derivatives thereof in the DNA were converted to deoxyribonucleosidic compounds, which were soluble in 75% ethanol, 50 mM histidine, 1 II’IM sodium azide at pH 9, and were recovered in the filtrate. The fluorescence of crosslinked compounds prepared by this method was not significantly decreased by heating to 45°C for 1 day. As previously reported for RNA or unmodified DNA (24), the digestion of iodinated DNA was optimal at about pH 6. However, optimal elution of the DNA from the nitrocellulose filters occurred at pH 8. Therefore, because the buffer used needed good buffering capacity at pH 6 as well as pH 8-9, histidine was chosen. The greatest percentage of the DNA was eluted from the filters by 35-60 mM histidine, and the concentration of the buffer did not significantly afIect digestion of the DNA by nuclease P, . For this reason, 50 mM histidine was chosen for elution and digestion.

AND TAPPEL

HPLC. When the ammonium phosphate used for HPLC was pH 5 or below, the retention times of all the labeled compounds were low and they were not separated satisfactorily. Separation was better at pH 7 than at any lower pH. Concentrations of buffer greater than 50 mM did not improve the separation; therefore, 50 II’IM was the concentration chosen for column elution. When the iodinated, crosslinked adducts were purified by HPLC, fluorescence could be measured by monitoring the eluate with about 60% of the sensitivity of manual measurement of the fluorescence of individual fractions. The amount of radioactivity in these fractions was that expected from measurement of fluorescence. However, in most of the studies in which the compounds were detected by radioactivity, there was not a sufficient amount for detection by fluorescence. The addition of methanol to the ammonium phosphate buffer decreased the retention times of IdC and the crosslinked compounds. Concentrations of methanol greater than 10% decreased the separation of these compounds significantly, but 5% methanol did not. Because the amounts of crosslinked adducts obtained from DNA damaged in vivo were expected to be 5-6 orders of magnitude less than that of IdC, 2% methanol was chosen as the optimal compromise for separation of the crosslinks from IdC and for speed of analysis. PIC A did not significantly affect the retention time of IdC-MDA-dG, but 5 IrIM PIC A approximately doubled the retention time of IdC-MDA-Lys in the presence or absence of 5% methanol. Higher concentrations of PIC A had no further effect on the retention time of IdC-MDA-Lys. When the 50 mM ammonium phosphate buffer, pH 7, contained 5% methanol and 5 mM PIC A and the Ultrasphere ODS column was used, the retention times of a mixture of IdC, IdCMDA-Lys, and IdC-MDA-dG were about 12, 30, and 47 min, respectively. When a lofold excess of IdC was used, it did not significantly interfere with measurements of

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MEASUREMENT

OF MALONDIALDEHYDE-DNA

radioactivity in the crosslinks. However, due to inadequate separation, a lOOO-fold excess of IdC made accurate determination of radioactivity in these adducts extremely difficult in the presence of 5% methanol. The separation of the crosslinks from IdC was nearly doubled by adding 2% methanol and 5 IBM PIC A to the ammonium phosphate buffer. The retention times of IdC, IdC-MDA-Lys, and IdC-MDA-dG were 12,48, and 78 min, respectively; when the Zorbax ODS column was used, the retention times were 27, 78, and 90 min, respectively. Flow liquid scintillation. The flow liquid scintillation counting system had a background of 30 cpm and a counting efficiency of 35% for iz51. It resolved peaks of radioactivity that were 2 min apart if they contained at least lo4 cpm. When the time constant of the ratemeter was 100 s, a peak that contained 15-20 cpm above background was detected if the peak had a width of 5 min or more. Thus, if the specific activity of the ‘*? were 10’ cpm/nmol, subpicomole quantities of the iodinated, crosslinked heterodimer were detected. Figure 2 shows a typical chromatogram of a mixture of IdC, IdC-MDA-Lys, and IdC06

TIME, MIN

FIG. 2. Flow liquid scintillation counting of HPLC separation of 6 nmol of base pairs of iodinated DNA that contained about 330 crosslinks per lo6 base pairs and purified IdC-MDA-Lys equivalent to 200 crosslinks per lo6 base pairs on a Zorbax ODS column. The time constant of the rate meter was 3 s for lo5 cpm full scale and 100 s for 500 cpm full scale. (A) IdC, (B) IdCMDA-Lys, and (C) IdC-MDA-dG.

CROSSLINKS

269

DNA BASE PAIRS,NMOL FIG. 3. Radioactivity measured by flow liquid scintillation counting as a function of the concentration of a standard mixture of IdC (0) (X10e3), IdC-MDA-Lys (m), and IdC-MDA-dG (A). Chromatographies were done in duplicate.

MDA-dG eluted from a Zorbax ODS column. The peaks of radioactivity were well separated and the areas under all three peaks could be accurately determined. The peaks at 22 and 34 min were also observed in blanks that were prepared by addition of ethanol to the reaction mixture, but not in blanks that were prepared by gel filtration. Sensitivity of detection was mainly a function of the specific activity of the 125I used in the various experiments. Figure 3 shows that the radioactivity measured by flow liquid scintillation counting was proportional to the amount of sample applied to the column. The average standard deviation was 4% and the correlation coefficient was 0.99. After flow counting, the fractions collected at times that corresponded to the retention times of the crosslinked adducts and that contained amounts of crosslinked adducts large enough to measure fluorescence had fluorescence spectra similar to those previously seen for DNA that was crosslinked by MDA (1). This fluorescence could be abolished by reduction with borohydride. These fluorescence results identify these compounds as crosslinked heterodimers (21). These heterodimers were not

270

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present in DNA that had not been crosslinked but that had been treated and analyzed as described. DISCUSSION

In 1968, Brooks and Klamerth found that MDA binds primarily to the guanine and cytosine residues of DNA (25). Further studies showed that the changes produced in DNA by reaction with MDA are characteristic of crosslinking (1). When rats were fed MDA for 2 weeks, their hepatic DNA template activity decreased to 50% of the control values (26). These results demonstrated that MDA can crosslink DNA in vivo. To better understand the role of crosslinking of DNA in the pathobiology of aging, disease, and damage by genotoxic agents, measurement of the amount of crosslinking is necessary. Isolation of crosslinks made by treating DNA with formaldehyde in vitro (27) and direct measurements of DNA-benzopyrene adducts produced in vivo (28) have been accomplished by enzymatic digestion of the DNA and analysis of the digests by HPLC. Reduction of the crosslinks with borotritiide (2 1) was a more sensitive method of measurement than the measurement of fluorescence, but was limited by high background counts that presumably were due to nonspecific base reduction. The method described above combines the resolving power of HPLC with the sensitivity of radiolabeling. Measurements are quantitative and reproducible. Furthermore, because IdC can be measured as well as the crosslinks, the extent of crosslinking of the DNA can be directly determined as the ratio of crosslinks to IdC. This ratio is independent of the sample size, the extent of iodination, the specific activity of the 1251,and the sensitivity of the counting method. However, the amount of DNAprotein crosslinking in vivo may be greater than that detected by this method because MDA can also crosslink the c-amino group of lysine to the amino groups of adenine and guanine.

AND

TAPPEL

One of the characteristics of DNA that has been crosslinked by MDA is its resistance to DNase I (1,26); therefore, this enzyme was not used. Attempts to analyze crosslinked nucleotide heterodimers produced by the digestion of crosslinked DNA with only nuclease P1 failed because many more labeled and/or fluorescent compounds were seen than could be explained if nucleotides were the only products. Nuclease P, has phosphomonoesterase activity (24), which may account for some of the heterogeneity of the products. Depurination of DNA during the iodination at low pH may also have formed more products (22); this problem was minimized by carrying out the reaction at pH 5. Complete dephosphorylation of the crosslinked adducts produced only one product when only interstrand crosslinks were formed. Because the retention time of the crosslinked adduct was affected by methanol but not by PIC A, it could be separated easily from IdC. The retention time of IdC-MDA-Lys was affected by PIC A, presumably because of the carboxyl group; it could, therefore, be adjusted independently of the retention times of IdC and IdC-MDA-dG for optimal separation of the three compounds. Psoralens interact with the cytosine residues of DNA (13) as do other agents that modify only single bases. Therefore, subsequent radiolabeling and digestion of the DNA by the procedure described above could provide a more sensitive assay for other kinds of damage to DNA if cytosine residues are attacked, although a different chromatographic procedure would have to be used. For the isolation of damaged DNA from bacteria or cells in culture, proteinase K could be used to expose all the DNA-protein crosslinks, as was done in the present study. Lipid peroxidation can be caused by a variety of agents (2-6), and it occurs in a variety of disease states. Damage to the DNA is of medical importance, especially damage done by crosslinking (lo- 12,17,29). Studies on the crosslinking of DNA in vivo by MDA generated from methyl ethyl ketone peroxide,

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OF MALONDIALDEHYDE-DNA

an inducer of lipid peroxidation, have been done using the methods described herein (8). Also, these methods have been applied in a study of crosslinking of DNA in liver and testes of rats that produced MDA in viva from a precursor, 1,3-propanediol(9). Further details of the optimization of the conditions of assay and further application to biological systems are described elsewhere (7). ACKNOWLEDGMENT This research was supported by NIH Grant AM-09933 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases.

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27. Chaw, Y. F. M., Crane, L. E., Lange, P., and Shapiro, R. (1980) Biochemistry 19, 5525-5531. 28. Boroujerdi, M., Kung, H.-C., Wilson, A. G. E., and Anderson, M. W. (1981) Cancer Res. 41, 951957. 29.

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