Chem. Res. Toxicol. 1999, 12, 979-984
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Comparison of 32P-Postlabeling and High-Resolution GC/MS in Quantifying N7-(2-Hydroxyethyl)guanine Adducts Ingvar Eide,*,† Chunyan Zhao,‡ Rajiv Kumar,‡ Kari Hemminki,‡ Kuen-yu Wu,§ and James A. Swenberg§ Statoil Research Centre, N-7005 Trondheim, Norway, Center for Nutrition and Toxicology, Department of Biosciences, Karolinska Institute, Novum, S-141 57 Huddinge, Sweden, and Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599 Received March 3, 1999
This study compares 32P-postlabeling and high-resolution gas chromatography/mass spectrometry (GC/MS) in the quantification of N7-(2-hydroxyethyl)guanine adducts (7-HEG) in DNA obtained from the same tissue samples of control rats and rats exposed to ethene. The samples were obtained from two independent studies. In one study, male Sprague-Dawley rats were exposed to 300 ppm ethene for 12 h/day for 3 days (“Euro samples”). In the other study, male F-344 rats were exposed to 3000 ppm ethene for 6 h/day for 5 days (“U.S. samples”). DNA from liver and kidney from the European study was isolated in the European laboratory, and DNA from liver and spleen from the U.S. study was isolated in the U.S. laboratory. The DNA samples were coded, divided into two portions, and exchanged between the two laboratories. All DNA samples from both laboratories were analyzed with respect to 7-HEG adducts by 32P-postlabeling and high-resolution GC/MS in the European and U.S. laboratories, respectively. However, the U.S. samples were repurified in the European laboratory before the postlabeling analysis. The data from the Euro and the U.S. samples were therefore treated separately in the regression analysis of the 32P-postlabeling versus GC/MS data. The slope of the regression line for the Euro samples was 1.19 (r ) 0.97), implying that the GC/MS data were slightly lower than the postlabeling data (one possible outlier was excluded). The slope of the regression line for the U.S. samples was 0.61 (r ) 0.94), implying that the GC/MS data were somewhat higher than the postlabeling data. The main conclusion from this study is that there is very good agreement between the 32P-postlabeling and high-resolution GC/MS methods in quantifying 7-HEG adducts to DNA, particularly when identical DNA samples are analyzed and the RNA content is 99.5% pure. Salmon testis DNA, proteinase K, pancreatic ribonuclease, and micrococcal nuclease were obtained from Sigma Chemical Co. (St. Louis, MO). Nuclease P1 and spleen phosphodiesterase were from Boehringer Mannheim (Mannheim, Germany), and [γ-32P]ATP and T4 polynucleotide kinase were from Amersham (Little Chalfont, U.K.). The polyethyleneimine cellulose TLC plates were purchased from Macherey-Nagel (Du¨ren, Germany). HPLC grade methanol and 1 mL Bakerbond anion-exchange cartridges were from J. T. Baker (Deventer, The Netherlands). All other chemicals were analytical grade and were obtained from Sigma or Merck (Darmstadt, Germany). Chemicals for the U.S. Laboratory and High-Resolution GC/MS. Ethene was purchased from Matheson Gas Products (Twinsburg, OH) and was >99.9% pure. Pentafluorobenzyl bromide (PFBBr), potassium hydroxide (KOH), tert-butylnitrite, toluene, and tetrabutylammonium sulfate (Bu4NHSO4) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Gas chromatography quality hexane, dichloromethane, and ethyl acetate were obtained from Baxter Diagnostic Inc. (McGaw, IL). The sources of DNA purification grade sterilized Dulbecco’s phosphate-buffered saline (PBS) lysis buffer [100 mM Tris (pH 8.0), 0.2 M NaCl, 0.5% N-lauroylsarcosine, 4 M urea, and 10 mM 1,2-diaminocyclohexane-N-tetraacetic acid], 70% phenol/ water/chloroform reagent, RNase T1, RNase A, and other DNA isolation and HPLC reagents have been described previously (6). Animals, Exposure, and DNA Isolation of Euro Samples. The inhalation study was carried out at the Institute of Pharmacology and Toxicology (NTNU, Trondheim, Norway) and has been described in detail previously (8). Male SpragueDawley rats were provided by Møllegaard A/S (Skensved, Denmark). At the start of the experiment, the weights of the animals ranged from 180 to 225 g. The animals were exposed to ethene in conically shaped 0.7 m3 steel chambers with glass front doors and walls. There were eight animals in each cage and a maximum of four cages in each inhalation chamber. The rate of air exchange was 15 per hour. The temperature and humidity were kept within limits of 22 ( 0.3 °C and 65 ( 5% relative humidity, respectively. The aimed concentration of ethene of 300 ppm was generated by introducing a controlled stream of pure gas delivered by a two-stage precision pressure regulator. Exposures were performed during the daytime for 12 h/day for 3 consecutive days. Control groups of animals were treated in a manner identical to that of the exposed groups except for the absence of ethene. The concentration of ethene in the inhalation chamber was monitored hourly by gas chromatography. The mean concentration ((SD) of ethene for exposure for 3 days was 302.1 ( 4.6 ppm. Immediately after the last exposure, the animals were removed from the chamber one at a time for immediate decapitation and sample preparation. Liver tissues (1.5-2 g from lobus sinister) and kidneys were transferred to glass vials and frozen at -80 °C.
Eide et al. DNA was isolated from cellular nuclei of livers and kidneys as described by Gupta (18). Briefly, the nuclear pellets were treated with RNase A and T1 in 50 mM Tris-HCl buffer (pH 8.0), followed by pancreatic ribonuclease and proteinase K treatment. Proteins were removed by phenol extraction followed by chloroform/isoamyl alcohol extraction. DNA was precipitated by ethanol and washed with 70% ethanol. The precipitated DNA was redissolved in water, the concentration measured by absorption at 260 nm with a spectrophotometer, and the level of RNA contamination (2% is not a problem when identifying 7-HEG adducts by GC/MS. On the other hand, although the standard deviations in control animals are small, they constitute a relatively large percentage of the mean values (relatively high coefficients of variation), implying that relative differences as seen in exposed animals between GC/MS and postlabeling data may be possible also in control animals. The consideration above implies that the data obtained by GC/MS for 7-HEG in U.S. samples are too high. An alternative explanation that cannot be ruled out is that the data obtained by postlabeling for 7-HEG in U.S. samples are too low due to the extra purification step, e.g., because some DNA adducts were removed. The results show that there is one possible outlier among the GC/MS data for the Euro samples. Removing
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it improves the correlation coefficient significantly. The possible outlier is shown in Figure 1, but it is not included in the regression line. The agreement between the two methods for the Euro samples then becomes excellent with a high correlation coefficient and a slope of ∼1. Furthermore, coefficients of variation in exposed animals are relatively small. Hence, the main conclusion from this study is that when identical DNA samples are analyzed and the RNA content is
