DNA Methylation in Rat Liver by Daminozide, 1, 1-Dimethylhydrazine, and Dirnethylnitrosamine
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f1.JNDAMENTAL AND APPLIED TOXICOLOGY 11, 723-7 30 ( 1988)' DNA Methylation in Rat Liver by Daminozide, 1, 1-Dimethylhydrazine, and Dirnethylnitrosamine PETER SAGELSDORFF, WERNER K. LUTZ, 1 AND CHRISTIAN SCHLATTER Institute ofToxicology, Swiss Federal Institute ofTechnology and University ofZürich, CH-8603 Schwerzenbach, Switzerland Received February8, 1988: accepted May 27, 1988 DNA Methylation in Rat Li ver by Daminozide, 1, 1-Dimethylhydrazine, and Dimethylnitros amine. SAGELSDORFF, P., LUTZ, W. K., AND ScHLAITER C. (1988). Fundam. Appl. Toxico/. 11, 723-730. [methyP4C]Daminozide (succinic acid 2',2'-dimethylhydrazide; 37 mgjkg), l,l (14C]dimethylhydrazine (UDMH; 19 mgtkg), and (14C]dimethylnitrosamine (DMNA; 0.1 mg/ kg) were administered by oral gavage to male Sprague-Dawley rats. After 24 hr, the animals were killed and DNA was purified from the livers to constant specific radioactivity. After enzymatic degradation of the DNA to the 3'-deoxynucleotides the Ievel of DNA methylation was deter mined by HPLC analysis. Radiolabeled 7-methylguanine (7mG) was identified by cochromatog raphy with unlabeled 7mG added as standard after acidic depurination of DNA and HPLC analysis ofpurines and apurinic acid. All three compounds were found to methylate DNA. The relative potencies were 1:47:4900 for daminozide:UDMH:DMNA. With [methyPH]UDMH, the formation of7mG was investigated as a function of dose administered, at 20, 2, and 0.2 mgj kg. The methylation ofDNA was strictly proportional to the dose. The data were used to com pare the Ievel of DNA alkylation derived from residues of daminozide and UDMH in treated apple with the genotoxicity of the intake of N-nitroso compounds in Germany and Japan. It is estimated that these residues could Iead to a DNA methylation in the Iiver of about 6% of an average exposure to DMNA. © 1988 Society ofToxicology. Daminozide (succinic acid 2'-2'-dimethylhy tumors of the lungs, kidneys, and, for drazide; trade name of formulation: Alar) is UDMH only, the liver. The studies are a plant growth regulator used in fruit and difficult to evaluate properly, however, be flower culture. Degradation of the parent cause of the high toxicity of the treatments compound in the fruits as weil as in mam (23 and 8% survival at 70 weeks). Since dami mals may, under certain conditions, yield nozide was much less potent than UDMH 1, 1-dimethylhydrazine (unsymmetrical di (by a factor of about 200}, it is possible that methylhydrazine, UDMH). The tumorigenic UDMH formed as a metabolite might be re potential ofvarious substituted hydrazine de sponsible for the effects induced by damino rivatives has been investigated, almost exclu zide. In rats, UDMH was a very weak liver sively in one laboratory. Both daminozide carcinogen, producing tumors only late in (2% in drinking water; Toth et al., 1977) and life. It was at least two orders of magnitude UDMH (0.01% in drinking water; Toth, less potent than dimethylnitrosamine 1973) have been reported to induce angiosar (DMNA) (Druckrey et al., 1967). comas in mice in various organs, as weil as The putative mechanism of a tumor induc tion by UDMH might involve DNA methyl ation, in analogy to the mode of action of 1 To whom correspondence should be addressed. the potent carcinogen 1,2-dimethylhydrazine 723 0272-0590/88 $3.00 Copyright© 1988 by the Society ofToxicology. All rights of reproduction in any fonn reserved. 724 SAGELSDORFF, LUTZ, AND SCHLATTER (symmetrical dimethylhydrazine, SDMH) in [methy/-'4C]UDMH at a limit ofdetection of0.006% 14 (Lewis and Swenberg, 1983). Oxidative de [methyi- C]DMNA. The initial radiochemical purity of (methyl-3H]VDMH (10 Ci/mmol) was 76%. This com methylation ofUDMH can, in principle, Iead pound was purified before use by HPLC on a Partisil 10 to the same metabolic intermediates, so that SCX (250 X 4 mm) column followed by a Partisil5 ODS a methylation ofDNA by UDMH cannot be 3 (250 X 4 mm) column (Whatman, NJ). eluting at a flow excluded. of 1.5 ml/min with a linear gradient over 20 min from 5 The data obtained from in vitro tests on the mM HCOOH/NH3 , pH 3.6, to 300 mM HCOOH/NH3 , 50% methanol~ pH 3.6. The retention times were 9 and genotoxicity of alkylhydrazines do not corre 28 min for DMNA and UDMH, respectively. The frac late with carcinogenicity (Kier et al., 1986), tions containing UDMH were pooled and lyophilized. probably because the activation to the ulti The radiochemical purity of the purified [methyPH] mate electrophiles does not proceed quantita VDMH was 97.7% and contained less than 0.05% 3 tively. It was therefore the aim ofthis study [ methyl- H]DMNA. Anima/s. Male Sprague-Dawley rats [Iva:SIV-50.SD] to measure the methylating potency of dami were obtained at weights of about 150 g from Ivanovas, nozide and UDMH in vivo. Using the Kissleg, FRG. They were held four per macrolone cage methyl-radiolabeled compounds, it was on sawdust for 1 week for accJimatization. They were fed tested to what extent daminozide and Haltungsdiät ''A" No. 343, Klingental Mühle AG. Kais UDMH methylate liver DNA in male eraugst, Switzerland a'd libttum and had free access to tap water. Sprague-Dawley rats after oral administra Treatments. The rats received the labeled test com tion. The results were used to estimate the pounds in about 1 ml 0.01 N HCI by oral gavage. [methy/- Ievel of DNA methylation induced by resi 3H]UDMH was diluted with unlabeled UDMH (Huka dues of daminozide and UDMH in fruits, AG, Buchs, Switzerland) to the appropriate doses. The and the DNA alkylation was compared with precise dose given to each individual animal was deter that arising from an average intake of dimeth mined on the basis ofthe weight difference ofthe syringe before and after oral gavage. The values are listed in the ylnitrosamine (DMNA) with foods (Preuss tables. Animals receiving the 14C Iabel were placed in mann and Eisenbrand, 1984). glass metabolism cages and the exhaled air was trapped with ethanolaminejmethanol ( 1 + 4 vol) to collect ex pired 14C02. MATERIALS AND METHODS Iso/ation ofchromatin. Twenty-four hours after the ad ministration, the animals were k.illed by open heart punc Test compounds. The radiolabeled test compounds ture under ether anesthesia. The livers were excised and daminozide, UDMH, and DMNA were obtained from homogenized in a Teflon Potter-Elvehjem-type homog Chemsyn Science Labs., Lenexa. Kansas. The radio enizer at 4°C. Chromatin was prepared according to Sa chemical purity of [methy/-14C]daminozide (20.35 mCi/ gelsdorff et a/. ( 1983), essentially by precipitation with mmol) was 97 .3%, as determined by HPLC on a Lichro the nonionic detergent Nonidet-P40 (BDH Chemieals Ltd., Poole BH 12 4NN, England). This pellet, containing sorb RP18 (7 ~m) column (4 X 250 mm), eluting at a · ßow of 1.5 ml/min wjth 65 parts MeOH and 35 parts about 2-3 mg DNA and 20-30 mg protein per gram 0.1% (w/v) aqueous SDS solution containing 0.1% (v/v) Ii ver, was washed until the suspension contained < 1 #'Ci formic acid. Fractions of 1 min were collected and total radioactivity in order to remove the majority of counted for radioactivity after the addition of 10 ml In noncovalently bound radioactivity. sta-Ge) (Packard). The retention time for daminozide Isolation ojDNA. These steps were perfonned accord was 6 min. The radiochemical purities of [methy!-' 4C] ingtoSagelsdorff et al. (1983). Essentially, thechromatin UDMH (17.3 mCi/mmol), [methyl- 14C]DMNA (17.3 pellet was homogenized in a Waring biender in 25 m1 mCi/mmol), and [methyl-3H]DMNA (10.8 Cijmmol) lysing medium (1% (w/v) SOS, 10 mM EDTA, 8 M urea were determined by HPLC on a Lichrosorb RP 18 (7 ~m) in 0.24 M sodium phosphate, pH 6.8). Protein was ex column (4 X 250 mm) eluting at a flow of 1.5 mlfmin, tracted from the homogenate with chloroform/isoamy1 with a linear gradient over 15 min of 10-50% methanol alcohol/pheno) (CIP), phenolwas removed with diethyl in 50 mM sodium phosphate buffer, pH 3.5, containing ether, and the DNA was purified by adsorption on a hy I 0 mM octanesulfonic acid. The retention times were droxylapatite column, dialysis, and precipitation with 3 and 9 min for DMNA and UDMH, respectively. The ethanol. The highly purified DNA (mostly double radiochemical purities were 99.8, 95.9, and 90% stranded, less than 0.2% protein, as shown with radiola for [methy/- 14C]UDMH, [methyl- 14C), and [methyl- beled amino acid incorporation [Caviezel et al.. 1984]) 3H]DMNA; no DMNA contamination could be detected was dissolved in a 20 mM sodium succinate buffer, con- DAMINOZIDE DNA METHYLATION 725 taining 8 mM CaCh, pH 6.0. The amount of DNA was dioactivity was determined by analyzing an inactive determined by assuming an absorbance of 20 at 260 nm DNA digest. The standard deviation for each fraction for a solution of 1 mg DNA/ml. Scintillation counting was determined on the basis of 17 nucleotide anaiyses of (Packard scintillation counter Tricarb 460 CD} was per control DNA hydrolysates. The standard deviation of a formed on an aliquot ofthe DNA solution after addition sample containing little radioactivity was assumed to be of I 0 ml lnsta-Gel. equal to the standard deviation of the background sam Repetitive purification of DNA. The remaining DNA ples. The Iimit of detection for radioactivity in a fraction solutionwas mixed with 25 mllysing medium, extracted was then calculated on the basis of2 SD. twice with CIP and ether, and dialyzed and precipitated Analysis of DNA purines. DNA (0.1-1 mg) in 1.5 ml with ethanol.