Incorporation of radioactivity from labeled di-(2-ethylhexyl)phthalate into DNA of rat liver in vivo

Chem.-Biol. Interactions, 44 (1983) 1--16 Elsevier Scientific Publishers Ireland Ltd.

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OF RADIOACTIVITY FROM LABELED DI-(2ETHYLHEXYL)PHTHALATE INTO DNA OF R A T LIVER IN VIVO INCORPORATION

PHILLIP W. ALBRO, JEAN T. CORBETT, JOANNA L. SCHROEDER and SANDRA T. JORDAN Laboratory of Environmental Chemistry, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.) (Received May 14th, 1982) (Revision received August:Sth, 1982) ~Accepted August 9th, 1982)

SUMMARY

Di-(2-ethylhexyl)phthalate (DEHP), when fed at high levels in the diet for two years, is reportedly an hepatocarcinogen to rats and mice. Radioactivity from ethylhexyl-labeled, but n o t from phthalate-labeled, [~4C]DEHP is associated with highly purified DNA from the livers of treated rats and this radioactivity is not accounted for by assumptions of adsorption, intercalation, attachment to R N A or histones, an impurity in the labeled DEHP, or artifactual binding during sample workup. Spontaneous binding of radioactivity to DNA from either ethylhexyl-labeled DEHP or its total urinary metabolites could n o t be detected. Although rat liver slices generated all of the known metabolites of DEHP in vitro, no binding to DNA occurred. Administration of dual 3H/'4C-labeled DEHP to rats yielded liver DNA whose 3H/14C ratio was inconsistent with the attachment of any reasonable multi-carbon fragment from the ethylhexyl portion to the DNA. The observation that roughly 100 times as high a percentage of the 14C administered was found in urea as in total DNA suggests that the ~4C entered DNA through carbamyl phosphate, a precursor of both urea and pyrimidine bases. If this is the case, the association of C-1 from the ethylhexyl portion of DEHP with DNA m a y not involve alteration of the DNA or genetic damage.

Key words: Di-(2-ethylhexyl)phthalate -- Covalent binding -- DNA -- Rats -Phthalate

Abbreviations: DEHP, di-(2-ethylhexyl)phthalate; GLC, gas-liquid chromatography; HPLC, high pressure liquid chromatography; MEHP, mono-2-ethylhexyl phthalate; TLC, thin-layer chromatography. 0009-2797/83/$03.00 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

INTRODUCTION DEHP is a ubiquitous environmental pollutant to which humans are exposed by a variety of routes. DEHP leaches into blood and blood products that are stored in PVC plastic bags [1--3] and is then transfused into humans receiving those products. DEHP is found in lakes and rivers at levels up to 300 ppm in water samples and as high as 71 000 ppm in the sediment [4], although average levels are much below these extremes. Since DEHP is the most c o m m o n plasticizer used in PVC and several other types of plastics, almost everyone has at least topical exposure to the compound. It is because of this nearly universal exposure to DEHP, especially high in the case of factory workers involved in the manufacture of PVC films [ 5], hemophiliacs [6] and hospital patients receiving either cardiopulmonary bypass or hemodialysis therapy [7], that concern about possible human health hazards has persisted for many years. Although the acute toxicity of DEHP is very low (LDs0 = 28--34 g/kg in the rat [8] ), liver damage [9,10] and testicular atrophy accompanied by reduced levels of testosterone [11] are associated with chronic exposure to high levels of DEHP in laboratory animals. Recently the National Toxicology Program [12] reported that continuous exposure of both rats and mice to a maximum tolerated dose (0.6--1.2% of the diet) of DEHP for t w o years resulted in a statistically significant increase in hepatocarcinoma in both species. At the same conference [12] we reported that radioactivity from [1-'4C]DEHP, but not from [7-14C]DEHP, was tightly associated with purified DNA from the livers of rats that had been administered the test compounds 1 or 10 days prior to sacrifice [13]. At that time it was impossible to state with any certainty whether or n o t the radioactivity was covalently b o u n d to the DNA. The extent of covalent binding of a test c o m p o u n d or its metabolites to DNA in vivo has been proposed as an indicator of potential carcinogenicity through genotoxic action [14]. Even if such binding occurs, however, it need not result in carcinogenesis. Depending upon the site and extent of binding, the lesion may be enzymatically repaired [15] or simply irrelevant to the carcinogenic process [16]. In the absence of covalent binding to DNA, a chemical substance may still contribute to the process of carcinogenesis through less direct mechanisms. Interference with DNA repair processes and inhibition of cell mediated immune surveillance are examples of indirect mechanisms by which a comp o u n d may promote t u m o r production whether or not it has a direct genotoxic activity [ 1 4 ] . The present study was performed in order to address the question of whether or n o t DEHP or its metabolites covalently bind to DNA in rat liver in vivo. The association of 14C with DNA reported previously [13] was a simple observation with many possible explanations, including intercalation, absorption, an experimental artifact, true covalent binding, or even that C-1 of ethylhexanol served as a precursor for normal DNA structures.

These various possibilities have been explored and evidence for and against them summarized in this report. MATERIALS AND METHODS

Materials (,4) Radiolabeled DEHP, mono-2-ethylhexyl phthalate (MEHP) and 2.ethylhexanol. DEHP labeled with ~4C in one of t h e carbonyl carbons was synthesized from [7-14C] phthalic anhydride (New England Nuclear, Boston, MA, 10.06 mCi/mmol) by esterification with 2-ethylhexanol as described previously [17]. Its radiochemical purity was at least 99.6% by radio-gas chromatography and radio-high pressure liquid chromatography (HPLC). These chromatographic techniques have been described in detail elsewhere [18]. DEHP labeled with ~4C at C-1 of the 2-ethylhexyl portions ([I'-~4C]DEHP), 18.4 mCi/mmol, was purchased from Pathfinder Labs., St. Louis, MO. As received and as used in the previous studies [13], its radiochemical purity was determined chromatographically to be only 95.1%, the main radioactive impurity being 2-ethylhexanol. This material was purified by preparative thin-layer chromatography (TLC) on silica gel with n-hexane/diethyl ether/ acetic acid ( 8 5 : 1 5 : 1, v/v). The product recovered after TLC was further purified by preparative reversed-phase HPLC using a Magnum 9, C-18 bonded phase column and an acetonitrile/water gradient from 80--100% in acetonitrile. The fraction containing the purified DEHP was diluted with 20 vol. of water and the DEHP extracted into n-hexane. The final radiochemical purity was greater than 99% (no impurities detectable by radio-GLC or radio-HPLC). MEHP labeled with ~ac at C-1 of the ethylhexyl portion was synthesized by enzymatic hydrolysis of the [I'-IaC]DEHP. This procedure, using nonspecific lipase purified from acetone powder of rat pancreas, has also been described previously [19]. Since the enzyme does not further hydrolyze MEHP [20], purification of the labeled MEHP is readily accomplished by partitioning between 0.4 M aqueous K2CO3 and diethyl ether/toluene (9:1) [18]. Any unreacted DEHP is removed in the organic phase, after which MEHP can be recovered by acidification of the aqueous phase. Saponification of [I'-~aC]DEHP to one part unlabeled phthalic acid plus two parts (molar ratio) 2-[1-'4C] ethylhexanol was described previously [13]. Dual-labeled DEHP was made by a procedure not previously described. Phthaloyl chloride, 205 mg (1 mmol), was allowed to react with 260 mg (2 mmol) of 2-ethyl-2-hexenol in 2 ml of dry pyridine at room temperature. The alcohol was obtained from Alfred Bader Chemicals, Metuchen, NJ and was found to be only 81% pure by gas-liquid chromatography (GLC) on 5% Carowax 20M at 125°C. The main impurities were 2-ethylhexanol (6.1%) and 2-ethyl-3-hexenol (9.7%), identified from their infrared, proton nuclear magnetic resonance (NMR) and methane chemical ionization mass spectra.

After the esterification products had been held overnight at 4°C, the reaction mixture was treated with 0.3 ml of water to hydrolyze unreacted phthaloyl chloride. The mixture was filtered through glass wool, diluted with 20 ml of diethyl ether, and washed with two 20-ml vol. of 0.4 M K2COa, 20 ml of water and 2 × 20 ml of 1 N HC1. The ether phase was dried over anhydrous Na2SO4, filtered and concentrated in a rotary evaporator to remove ether. The residue, in n-hexane, was chromatographed on 30 g of 60--100 mesh Florisil (partially deactivated with 5% water), eluting with 20 ml of hexane followed by 100 ml of hexane/diethyl ether (9:1, v/v). The desired product eluted in the second 50 ml of hexane/ether with no detectable contamination by DEHP (which eluted in the first 50 ml of hexane/ether) or alcohol (which was retained on the column). GLC of the product on a 2 m × 2 mm column of 3% OV-3 on 100--120 mesh Gas Chrom Q at 200°C revealed that it consisted of two significant components, one 76% and the other 22% of the total. Both components gave qualitatively identical electron impact and methane chemical ionization mass spectra [21] indicating that they were isomers. Chemical ionization mass spectrometry with isobutane as reagent gas confirmed that both compounds had a molecular weight of 386. Proton NMR and infrared spectra suggested that the major component was di-(2-ethyl-2-hexenyl)phthalate and the minor component was mono-2-ethyl-2-hexenyl, mono-2-ethyl-3hexenyl phthalate. Detailed analysis and discussion of the spectral data are beyond the scope of the present paper and wiil be presented elsewhere. A 3-ml glass vial was charged with 6 mg of platinum oxide (Adam's catalyst), a micro stir bar, 0.5 ml of n-heptane and 20 mg of the unsaturated phthalate diester. This was placed inside a heavy-wall 12 cm 3 bottle, with 15 mCi of powdered sodium [3H]borohydride (347.8 mCi/mmol, New England Nuclear) in the bottle but outside the vial. The bottle was sealed with a silicone rubber septum stopper and evacuated through an inserted syringe needle. The vacuum line was removed and 100 ~l of 33% acetic acid was injected through the septum onto the NaB3H4 to release tritium gas. The PtO: immediately turned black, indicating uptake of the reagent gas. The reaction mixture was stirred at room temperature for 4 h (magnetic stirrer), with sufficient vigor to keep the platinum black suspended. The reaction mixture was filtered through a 2-cm layer of anhydrous Na2SO4 above a tight plug of glass wool in a Pasteur pipet. The vial was rinsed with two 1-ml portions of n-pentane, which were also passed through the Na:SO4, followed by a final rinse with 2 ml of n-pentane. The filtrate was evaporated under N2 to 0.5 ml. Preparative TLC on Silica Gel GF in hexane/diethyl ether (85:15, v/v) eliminated hydrolysis/hydrogenolysis products and the material chromatographing at the Rf-value of DEHP standards run on the same plate (Rf = 0.74) was recovered. Final purification was by preparative HPLC using a SpectraPhysics model 8000B instrument, a Regis ODS-Sperisorb column (25 × 4.6 cm, 0.3 mmol bonded phase per g packing), with a solvent program starting at 85% acetonitrile, 15% water for 700 s changing to 100% acetonitrile for 600 s, at a constant

flow rate of 2 ml/min. Column temperature (room temp.) was 22.8°C. Under these conditions the elution times for standards were: 2-ethylhexanol, 148 s; di-(2-ethyl-2-hexenyl}phthalate, 344 s; DEHP, 494 s. The two unsaturated isomers of di-(ethylhexenyl)phthalate were not resolved under these conditions, so the (saturated) DEHP could be collected free of its precursors. The final radiochemical purity was found to be 99.2% by radio-GLC on OV-3. For the dual-label experiments, [3H] DEHP made as just described was mixed with [1 '-14C] DEHP to give the desired isotope ratio. Repetitive TLC of such a mixture on Silica Gel GF showed retention of the starting ratio (3H/14C), indicating that the positions labeled with 3H were n o t easily exchangeable under hydrogen bonding conditions. One may calculate that the [3H]DEHP should have had approx. 50% of its 3H at position 3 of the ethylhexyl side chains, 39% at C2 and 11% at C4, based on the isomeric composition of the starting di-(2-ethylhexenyl)phthalate and both the extremely low selectivity coefficient and minimal tendency to p r o m o t e double bond migration of platinum catalysts [22]. However, the incorporation of some 3H into positions other than 2, 3 and 4 cannot be ruled out. (B) Enzymes and specialty chemicals. Non-specific lipase was purified from acetone powders of rat pancreas as described previously [20]. Ribonuclease D, grade IV, was from Miles Laboratories, Elkart, IN. Pronase (ex. Streptococcus griseus), lot 030126, was from Calbiochem-Behring Corp., La Jolla, CA. Urease from Jackbean meal was Type VII from Sigma Chemical Co., St. Louis, MO. The urea used for isolation of DNA was ACS Certified grade from Fisher Scientific Co., Raleigh, NC, as were the sodium and potassium phosphates used for buffers. Sodium dodecyl sulfate (L4509) and calf thymus DNA {Type I) were from Sigma Chemical Co., St. Louis, MO. Type HTP hydroxylapatite was from Bio-Rad Laboratories, Richmond, CA. Cellulose pulp was Schleicher and Schuell No. 389. Non-radioactive DEHP, 99.6% pure, was donated by the National Toxicology Program [13]. Aquasol and Riafluor, media for liquid scintillation counting, were from New England Nuclear, Boston, MA.

Methods (A) Treatment of animals. Rats used in these studies have included CD strain males (Charles River Breeding Labs.) weighing 3 0 0 - 3 5 0 g, male Fischer rats weighing 120--130 g and female Fischer rats weighing 1 5 7 196 g. Radiolabeled phthalate or 2-ethylhexanol was given by gavage in 0.2 ml of cottonseed oil (peroxide value < 0.004, no detectable aflatoxins), after which the rats were allowed free access to NIH 31 chow containing 1% non-radioactive DEHP (w/w) and water for 24 h. Administration of the labeled test compounds was always at 09:00 h. In all cases the amount of 14C given was 100 ~Ci per rat. Urine was collected from rats held in metabolism cages and stored frozen at - 2 0 ° C until used. Metabolites of DEHP were extracted from urine and

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purified as described previously [17] except that they were not esterified with diazomethane. Rats were sacrificed by Nembutal overdose for isolation of liver DNA. (B) Isolation o f macromolecules. DNA was isolated from rat liver as described by Markov and Ivanov [23] with the following modifications: (a) all isolations started with a 1-g portion of liver; (b) the extractant mixtures were centrifuged at 20°C because the upper phase solidifies at 4°C; (c) hydroxylapatite columns were eluted with the assistance of centrifugation using Gelman 4662-G10 centrifuge filters. The hydroxylapatite was supported on glass fiber filter paper (Whatman) of 1-~m mean pore size. Fractions (5-ml) were monitored at 260 nm to determine when the elution of a given material was complete, to optimize solvent changes. The A ~ for single-stranded DNA was taken to be 292.5 at 260 nm and for doublestranded DNA the value of 206 was used. In our hands the procedure of Markov and Ivanov gave DNA whose UV absorbance ratios (260 n m / 2 8 0 nm and 260 nm/230 nm) averaged 2.08 + 0.057 and 2.19 + 0.10, respectively (17 rats). In some cases the DNA was further purified. DNA (2 mg), in 2 ml of 0.1 M NaC1 containing 0.05 M EDTA, 0.05 M Tris--HC1 (pH 7.4) at 37°C and 0.5% sodium dodecyl sulfate, was subjected to equilibrium dialysis overnight at 4°C against 50 ml of 1% sodium dodecyl sulfate. Both the dialysin and dialysout were freeze-dried, dissolved in distilled water and radioassayed in Aquasol. Prior to dialysis, some samples of DNA (2 mg portions) were incubated at 37°C with either 100 pg of Pronase or 50 pg of Ribonuclease for 16 h in the above buffer. Under these conditions the Pronase was shown to be able to make dialyzable up to 860 ~g of total rat liver protein (isolated from the phenol phase of the Markov and Ivanov procedure by precipitation with acetone). Other samples of radiolabeled DNA previously isolated according to Markov and Ivanov were repetitively extracted with acetone: 1 N HC1 (3 : 1, v/v), 1 ml/mg DNA, to remove any radioactivity that might be simply adsorbed (hydrogen bonded) or with 2 ml of 0.25 N aqueous HC1 to extract histones. The DNA recovered in these procedures was quantitated by the thiobarbiturate assay of Gold and Shochat [38]. In order to test for possible binding by intercalation, labeled DNA isolated from the hydroxylapatite column (double-stranded DNA) was heat-denatured in 5 ml of MUP buffer [23] at 100°C for 5 min. Hydroxylapatite, 0.5 g, that had previously been boiled in 5 ml of 0.125 M potassium phosphate (pH 6.8), cooled and centrifuged, was added to the denatured DNA, stirred and centrifuged. Any residual double stranded DNA was thus removed [ 2 4 ] , after which the supernatant, containing single-stranded DNA, was dialyzed against two 2-1 changes of water for 24 h. The dialysin was freezedried and radioassayed. All known metabolites o f DEHP [21,25] are dialyzable. (C) In vitro experiments. Portions (l-g) of rat liver were used to make tissue slices as described by EUiott [26]. 8ubstrates, either [l'-14C]DEHP

or a mixture of [I'-'4C]DEHP, [I'-'4C]MEHP and 2-[1.14C]ethylhexanol, were coated on the b o t t o m s of beakers or, in some cases, 12-ml bottles. Tissue slices (1 g) were incubated at 37°C in Krebs-Ringer bicarbonate buffer with the radiolabeled substrates for 1 h under 95% O~/5% CO2 with gentle swirling (rotary shaker). Those incubations performed in bottles involved a continuous flow of O2/CO2 through a septum stopper pierced with syringe needles, the efflux being washed through 30% ethanolamine in methyl cellosolve to trap radioactive 14CO2. After incubation the total incubation mixtures were either (a) processed for the isolation of DNA [23] or (b) extracted as described by Folch et al: [27] to recover the organic-soluble metabolites. The chloroform-methanol extracts were washed with 0.1 N HC1 [27] and aliquots of the aqueous and chloroform phases radioassayed in BBOT [28]. The chloroform phase was concentrated to remove solvent (rotary evaporator) and treated with etherial diazomethane for H P L c analysis of the metabolites as described in detail elsewhere [18]. In some cases the rinsed tissue slices and incubation medium were extracted and analyzed separately. Radioactivity b o u n d to the insoluble tissue residue was determined by digestion with NCS and radioassay in Aquasol. Calf t h y m u s DNA was dissolved in 12.5 mM NaC1 containing 5 mM sodium acetate (pH 5), at a concentration of 1 mg/ml. Portions (2-ml) of this solution were incubated at 23°C for 6 h with gentle swirling on a rotary shaker after the addition of 0.4 ml of methanol containing either 1 pCi of [1'-14C] DEHP or 1 ~Ci of total, urinary metabolites of [1'-14C] DEHP. After incubation, enough solid NaC1 was added to give 0.15 M and the DNA was precipitated with 2 vol. of absolute ethanol. Zero-time controls were also run. This procedure is essentially similar to that used by Kriek to study the binding of N-acetoxy-AAF to DNA [29]. The precipitated DNA was washed four times with 2-ml portions of acetone: 1 N HC1 ( 3 : 1 , v/v) to remove adsorbed radioactivity, aliquots of each wash were radioassayed in Aquasol. The final DNA pellet was dissolved in 1 ml of Digestol (Yorktown Research, New Hyde Park, NY) and radioassayed in Aquasol). (D) Urea. Urea was isolated as the dixanthate from 0.2-ml portions of the 24-h urine of rats previously given dual-labeled [3H/'4C] DEHP and crystallized from glacial acetic acid [ 3 0 ] . The crystals were dissolved and radioassayed in BBOT scintillation fluid [28]. Since the xanthydrol procedure is not totally specific, urine samples were also incubated at room temperature with and without urease in Conway diffusion cells [31]. Urine (0.25 ml) was placed in the center well and treated with either 50 ~l of 50 mM potassium phosphate (pH 7.4) or with 4 units of Jackbean urease in the same volume of buffer. The outer well contained 2.0 ml of 1 N KOH. After 2 h at room temp., 150 ul of 6 N HC1 was added to the center well through a rubber septum in the cover of the cell to release CO2 from the ammonium carbonate/carbamate. Diffusion was allowed to proceed for 2 h (more than enough time [31] ) after which a 0.1-ml aliquot of the KOH solution was

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radioassayed in BBOT. Saturated ag. BaC12 was added to the remaining KOH solution to precipitate BaCO3. The precipitate was collected on Whatman #541 paper and washed repeatedly with methanol, acetone and water until no 3H could be detected in the washings. The dual label used in these experiments thus provided a means to insure that only 14CO2 contributed to the final radioactivity. The BaCO3 was digested with 6 N HC1 and the released 14CO~ trapped in a 30% solution of ethanolamine in methyl cellosolve for radioassay in Aquasol. Radioactivity recovered in the absence of urease was subtracted from that recovered in its presence. This experiment was repeated three times. (E) Dual label experiment: DNA. Two female Fischer F344 rats weighing 157 g and 174 g, respectively, were each given, by gavage, 0.2 ml of cottonseed oil containing 100 uCi of di-(2-ethyl-[1-14C]hexyl)phthalate and 148 pCi or 241 t~Ci of [3H] DEHP, a total DEHP mass of approx. 2.4 mg. The rats were held in metabolism cages with free access to NIH 31 chow (containing 1% unlabeled DEHP) and water for 24 h, the urine (collected over a t h y m o l crystal to inhibit bacterial metabolism) was frozen and the rats were sacrificed. DNA was isolated from several 1-g portions of liver [23] and 1/10 of each DNA sample assayed for quantity of DNA and UV spectral properties. The remaining 9/10 of the DNA was radioassayed in Riafluor. The technique used for simultaneous measurement of 3H and ]4C has been clearly described [32], but several precautions were taken in the present study to ensure reliability of the radioassay results. Samples of the feeding solution (3H/'4C-DEHP in cottonseed oil) were diluted to give the same number of cpm in the ~4C channel of the Packard Tri-Carb Scintillation Counter as the DNA samples. These feeding solution samples were radioassayed before and after adding 2-mg portions of calf t h y m u s DNA to the solutions, to test for possible quenching of either the 3H or ~4C counts. No such quenching could be detected. Assaying the initial [3H/]4C]DEHP at the same ~4C level as the DNA ensured that linearity of the photomultiplier in the scintillation counter would not affect the apparent isotope ratios, and t h a t the standard error would be the same for both types of samples. Urine samples from these rats and reference standards (3H-H20, [14C]toluene) were similarly diluted to the same levels of radioactivity as the DNA samples. In all cases the amounts of DNA radioassayed were ~<2 mg and the external standard counting ratio indicated a constant counting efficiency throughout. Each sample was counted a minimum of 9 times, 10 min each time, one count per cycle through the samples. This procedure compensates for any drift in counting efficiency with time. Background corrections were made on the basis of scintillation fluid-solvent blanks corresponding to the appropriate diluents-cottonseed oil, water, or saline. No counts above background were detected in the ~4C channel when SH standards were assayed. Ratios (3H/14C, dpm) were compared for differences using 2-tailed t-tests. Samples recounted 72 h later had not changed. Standard curves for dilutions of feeding solution ranging from 1/20 to

20 times the cpm of the DNA samples were linear (linear correlation coefficients > 0.999 for both 3H and 14C). (F) Dual label experiment: urinary metabolites. The isolation of individual metabolites of DEHP from urine by preparative HPLC on S5CN (direct phase) and RP-8 (reversed phase) columns has been described previously [18]. Metabolites isolated from the urine of rats given the dual labeled DEHP (3H/14C initially 1.48) in the present experiment included 2-ethylhexanoic acid, mono-2-ethyl-3-carboxypropyl) phthalate (metabolite I), mono-2-ethyl-5-carboxypentyl phthalate (metabolite V), mono-2-ethyl-5ketohexyl phthalate (metabolite VI), mono-2-hydroxyethyl-hexyl phthalate (metabolite VII), mono-2-ethyl-5-hydroxyhexyl phthalate (metabolite IX) and mono-2-ethyl-6-hydroxyhexyl phthalate (metabolite X). Ratios of 3H/14C in these individual metabolites were determined as for the DNA above. RESULTS

A single pool of DNA from F344 female rats previously dosed with [I'-~4C]DEHP was treated in several ways to attempt to remove ~4C not covalently bound to the DNA. The results are summarized in Table I. All treatments resulted in an increase in the specific activity of the recovered DNA, even though some radioactivity and some of the DNA were always lost as a result of the treatment. In general, lengthy incubations followed by SDS dialysis led to poor recoveries of DNA. DNA isolated from the livers of F344 rats given the original 'as received'

TABLE I SPECIFIC ACTIVITY OF DNA AFTER VARIOUS TREATMENTS DNA Preparation

d p m / m g (+S.E.)

% Recovery a

From hydroxylapatite b A f t e r SDS dialysis After pronase A f t e r 0.25 N HCI A f t e r a c e t o n e HC1 Single-stranded D N A After ribonuclease

191.7 253.6 295.0 291.4 220.3 234.9 212.5

-66.0 58.3 63.4 75.9 64.6 35.7

-+ 9.6 + 7.2 ± 6.9 ± 6.5 + 7.4 ± 2.6 ± 12.8

a p e r c e n t a g e o f D N A t r e a t e d t h a t was r e c o v e r e d after treatment. b D o u b l e - s t r a n d e d D N A isolated, f r o m liver o f rats pretreated with [ 1 ~-~4C]DEHP, according t o p r o c e d u r e o f M a r k o v a n d I v a n o v [ 2 3 ] . P o r t i o n s s u b j e c t e d t o e q u i l i b r i u m SDS dialysis [ 3 3 ] b e f o r e or a f t e r a 16-h i n c u b a t i o n w i t h p r o n a s e or r i b o n u c l e a s e . O t h e r p o r t i o n s e x t r a c t e d w i t h acidic a c e t o n e or a q u e o u s HC1 [ 3 4 ] . Single-stranded D N A isolated f r o m t h e d o u b l e - s t r a n d e d D N A a f t e r h e a t - d e n a t u r a t i o n a n d r e - c h r o m a t o g r a p h y o n h y d r o x y l a p a t i t e [ 35 ].

10 [l'-14C]DEHP had a mean specific activity of 194.7 + 14.8 dpm/mg of DNA (4 preparations). DNA from the livers of F344 rats given the highly purified [1'-14C] DEHP had a mean specific activity of 259.4 + 5.2 d p m / m g of DNA (2 preparations). There was no reason, therefore, to suspect that the labeling of the DNA reported previously [13] might be due to impurities in the DEHP. This will be discussed further in a later section. The radioactive products generated when a mixture of [l'-14C]DEHP (36.5% of the initial 14C), [l'-14C]MEHP (37.2% of the initial radioactivity), and 2-[1-~4C]ethylhexanol (26.3% of the initial ~4C) was incubated in Krebs-Ringer bicarbonate buffer with slices of liver from untreated rats are summarized in Table II. Although all of the known metabolites of DEHP including COs were generated under these conditions, no detectable radioactivity was incorporated into DNA. We were also unable to detect any radioactivity above background associated with calf thymus DNA incubated directly for 6 h with either [1'-~4C] DEHP or its total urinary metabolites. The previous observation that DNA isolated from the livers of rats given 100 uCi doses of [7-14C] DEHP did not become radiolabeled [13] has been confirmed in both CD and F344 rats. All data to be presented below were derived from rats that had been given the dual-labeled [3H/14C]DEHP. The urea, crystallized as the dixanthate from the 24-h urine of one of these rats, contained 0.585 + 0.022% of the total ~4C in the urine (3 determinations, mean + S.D.). The more specific

T A B L E II P R O D U C T S F R O M I N C U B A T I O N O F [ l ' - 1 4 C ] D E H P A N D ITS H Y D R O L Y S I S P R O DUCTS WITH RAT LIVER SLIDES a Fraction

% of Substrate a

% of Products b

Intact DEHP MEHP c 2-Ethylhexanolc CO s MEHP metabolitesd E t h y l h e x a n o l metabolites Bound, insoluble

36.5 37.2 26.3 --

13.4 29.9 0.64 6.87 18.1 19.7 0.75 1.06 9.56 Of

Aqueous

-e

phase

Lost on handling DNA from hydroxylapatite

--

---

---

aLiver slices in Krebs-Ringer bicarbonate incubated under 9 5 % 0 2 - 5 % C O 2 for 1 h at 37°C with a mixture of [I'-'4C]DEHP, [I'-'4C]MEHP and 2-[l'-14C]ethylhexanol. bpercentage of '4C initiallysupplied (1 ~Ci). CHydrolysis of D E H P during incubation would have replaced some of the M E H P and 2~thylhexanol as they were metabolized. dAll metabolites stillretaining the phthalate moiety. See Ref. 21 for structures. eAll metabolites ('4C-labeled) lacking the phthalate moiety, except C O 2. See Ref. 25 for structures. f N o r a d i o a c t i v i t y a b o v e b a c k g r o u n d d e t e c t a b l e in 2 m g o f D N A .

11 urease procedure in which the released 14CO2 was trapped in aqueous KOH, precipitated as Ba~4CO3, washed, re-released with acid and re-trapped as ethanolamine carbonate, indicated that 0.55 +- 0.12% of the total ~4C in this urine was in urea. The t w o methods agreed remarkably well, considering the number of manipulations required in the urease procedure. The 3H/~4C ratios measured for individual metabolites of di-(2-ethy!-[1-~4C; 2,3,4-3H]hexyl)phthalate isolated from the urine are listed in Table III. Six of the metabolites, V, VI, VII, IX, X and 2-ethylhexnoic acid, had 3H/14C ratios not significantly different (mean = 1.46 -+ 0.024 S.D.) from that of the dual labeled DEHP initially administered (1.48 -+ 0.072 S.D.). One metabolite (I) had a 3H/~4C ratio of 1.33 -+ 0.035, which was significantly different from 1.48 (P < 0.005). The relevance of these observations will be discussed later. Table III further summarizes the results of t w o experiments in which duallabeled [3H/14C] DEHP, at a different 3H/14C ratio in the t w o cases, was given to female F344 rats. The total 24-h urine had the same 3H/'4C ratio within experimental error as the starting DEHP in Expt. 1, but the DNA had a much lower ratio. Similar results for the DNA were seen in Expt. 2, in which a higher 3H/~4C ratio for the starting DEHP was used. For the combination of 2 experiments, the 3H/~4C ratio in the DNA fraction from hydroxylapatite

TABLE III 3H/14C RATIOS 24 h A F T E R FEEDING [3H/14C]DEHP Expt.

Material radioassayed

3H/14C

±S.E.M.

N

1

Feeding solution a Total 24 h urine DNA from hydroxylapatite [ ~4C]Toluene (standard) 2-Ethylhexanoic acid b Metabolite I b Metabolite Vb Metabolite VI b Metabolite VII b Metabolite IX b Metabolite X b

1.48 1.40 0.23 0.017 c 1.43 1.33 1.46 1.48 1.42 1.48 1.45

0.016 0.032 0.021 0.018 0.014 0.018 0.015 0.015 0.014 0.015 0.015

20 10 13 13 2 4 5 2 2 2 2

Feeding solution a DNA from hydroxylapatite DNA after pronase Total liver protein ¢1

2.41 0.323 0.311 1.39

0.029 0.020 0.014 0.009

7 15 9 9

aThe feeding solution consisted of approx. 2.4 mg of di-(2-ethyl-[1'-14C; 2,3,4-3H]hexyl)phthalate in 0.2 ml of cottonseed oil and was administered by garage 24 h prior to sacrifice. bMetabolites isolated from the 24 h urine bY HPLC [18 ]. For structures s e e t e x t . CNot significantly different from zero. dThe mean 14C specific activity of liver protein was 428 dpm/mg.

12 was 14.4 + 1.0% of that in the DEHP. This does not correspond to the 3H enrichment at any of the labeled positions of the ethylhexyl moieties. The specific activity of the DNA relative to 14C was 190.0 + 5.7 dpm/mg DNA in Expt. 1 and 202.8 + 7.8 (S.E.M.) dpm/mg DNA in Expt. 2, comparable to that typically seen with [1'-14C] DEHP alone in F344 rats. The difference between the 3H/~4C ratio in the starting DEHP and that in the DNA fraction was highly significant (P < 0.001). Similarly, the 3H/~4C ratio in the DNA fraction was significantly different from zero (P < 0.001). Treatment with pronase did not change the 3H/~4C ratio of the DNA significantly in Expt. 2. Since the liver protein had a higher ~H/14C ratio than the DNA (though not as high as the feeding solution), the majority of both the 14C and 3H in the DNA fraction was apparently associated with DNA. DISCUSSION

Having repeatedly observed the association of radioactivity derived from ethylhexyl-labeled DEHP with DNA isolated from the livers of rats exposed to the labeled DEHP in vivo, we initially considered six possibilities to explain the phenomenon. The apparent binding to DNA might be an experimental artifact in the sense of occurring during sample workup. The binding may occur in vivo, but be due to a labeled impurity in the [1'-~4C] DEHP. The binding might not be to DNA, b u t instead to traces of protein (e.g. histone) or R N A carried through the purification procedure. The labeled carbon atom (C-1 of the 2-ethylhexyl part of DEHP) might enter the regular anabolic pathway for synthesis of normal DNA constituents. DEHP, MEHP or another metabolic product might be capable of spontaneously reacting with DNA. Or finally, an activated intermediate formed in the course of DEHP metabolism might be able to bind covalently to DNA. In the previously reported study [13] we observed that feeding a high level of unlabeled DEHP (1% of the diet) to rats for 10 days following a single oral dose of [1'-14C]DEHP completely eliminated our ability to detect any unbound, organic-extractable radioactivity in the livers. However, radioactivity associated with DNA was still easily detected. This indicated that the DNA-associated radioactivity was not exchangeable. It also left no obvious source of radioactivity from wlzich we could have generated an experimental artifact. In the present study we found that the DNA from various portions of a given liver had identical specific radioactivities within experimental error and that animal-to-animal variation within a group of similarly-treated rats did not exceed an RSD of +21%. Thus we have no reason to suspect the intervention of an experimental artifact. The data in Table I indicate that there was in these experiments 14C b o u n d to DNA that could not be accounted for as being due to adsorption, intercalation, association with histones or association with RNA. That the specific radioactivity of the residual DNA increased after each treatment may have significance, but the absolute value of the increase is not quantitatively

13 interpretable. There is no reason to assume that the 14C was distributed uniformly throughout the DNA; the failure to recover 100% of the starting DNA after the various treatments m a y have contributed to the extent of the changes in specific activity. Although chromatography of native DNA on hydroxylapatite is relatively independent of the length of the doublestranded chain [35], the same would not necessarily apply to SDS dialysis, acid precipitation, etc. Pronase is known to produce 'nicks' in doublestranded DNA chains even when DNase activity is inhibited by EDTA and SDS [36]. Treatment with acid and/or with heat may have caused some depurination under these conditions [37]. For these reasons we make no attempt to apply quantitative interpretations to the data in Table I. However, even if the DNA suffered some damage by the treatments summarized in Table I, the absence of 14C covalently bound to the DNA should have resulted qualitatively in a decrease in specific activity, and such did n o t occur.

Incubation of DNA with either [l'-14C]DEHP or its urinary metabolites did not result in binding o f 14C to the re-isolated DNA. We could have detected the binding of one ~4C-labeled, DEHP-derived carbon atom per 2 × 106 DNA bases in these experiments (radioactivity signal: background greater than 2.0), so spontaneous binding, if any, would have had to be below this level. As indicated in the Results section, highly purified [1'-~4C] DEHP appeared to be a somewhat more efficient precursor of DNA-associated radioactivity than was the 'as received', 95% pure material. This is n o t surprising since 2-[|4C]ethylhexanol, the main impurity in the original [I'-~4C]DEHP, was previously shown n o t to be effective at labeling liver DNA in vivo [13]. Incubation of neither [1'-14C] DEHP, [I'-~4C]MEHP nor 2-[1':~4C] ethylhexanol with rat liver slices in vitro caused ~4C to become b o u n d to DNA, in spite of the fact that all of the previously identified metabolites of DEHP were produced by the system. The quantitative data in Table II indicate that a b o u t 63% of the DEHP was hydrolyzed, about 99% of the 2-ethylhexanol, including that generated from hydrolyzed DEHP, was oxidized and that roughly 50% of the MEHP was further metabolized in this experiment. Metabolites of MEHP tended to stay physically inside the tissue (or adsorbed to it), while metabolites of 2-ethylhexanol tended to leak out into the medium. Since the incubation was run in bicarbonate buffer in an O2/CO2 atmosphere, the ~4CO2 released by metabolism would have immediately been diluted to a low specific radioactivity, one major difference between the in vivo and in vitro situations. Since the 2-ethylhexanoic acid isolated from the urine after administration of dual-labeled DEHP had the same 3H/14C ratio as the starting DEHP, it is clear that C-1 of the ethylhexyl chain of the DEHP did n o t contain 3H. A similar argument applies to the methyl carbons and C-5, since metabolites V, VI and VII retained the 3H. Accordingly, there is no reason to suspect that tritiation of the double bonds of di-(2-ethylhexenyl)phthalate under the conditions described here led to randomization of the label. The

14 3H/14C ratios for the DEHP metabolites and total urine (which contained roughly 50% of the label administered to the rats) shown in Table III indicate that in general the 3H did not significantly exchange out of the DEHP. The 3H/'4C ratio of 1.33 for metabolite I indicated that 10.1 + 0.34% of the original tritium was lost during its formation. This metabolite has lost C-5 and C-6 of the ethylhexyl chain, and has lost the protons originally attached to C-4. The observed 3H/'4C ratio, then, is in good agreement with the original estimation that 11% of the 3H in the dual labeled DEHP was at C-4 of the ethylhexyl chain. It is thus difficult to account for the 3H/~4C ratio observed for the DNA in these experiments, 14% of that for the starting DEHP. Clearly, however, the intact 2-ethylhexyl side chain is not simply attached to DNA, and the data in Table III do not support the assumption that even a t w o carbon unit including C-1 of 2-ethylhexanol serves as the labeling precursor for the DNA. The 3H/14C ratio in the DNA was less than half of what such an assumption would require (39%). Since C-2 of the ethylhexanol carries only one proton, one should lose either none or all of the 3H at that position during its metabolism. The amount of ~4C associated with the total DNA from the liver of an F344 rat represented only about 0.004% of the ~4C supplied to the rat as [I'-~4C]DEHP. In contrast, about 0.5% of the 14C was excreted as the carbon atom of urea. Since C-1 of 2-ethylhexanol does appear as '4CO2 both in vitro and in vivo [ 2 5 ] , it is reasonable to presume that label reached urea via its precursor carbamyl phosphate [39]. Carbamyl phosphate, however, is also a precursor of C-2 of the pyrimidine bases [39]. If this much label ends up in urea, it would be very surprising if 1% as much did not find its way into DNA precursors through the competing pathway. In vitro, such incorporation w o u l d be made indetectable by the diluting effect of the relatively massive amount of bicarbonate in the buffer. Failure of orally administered 2-[1-14C] ethylhexanol to provide label to liver DNA [13] may reflect its facile metabolism by alcohol dehydrogenase [25] throughout the body, and the likelihood that its tissue and subcellular distribution differs from that of DEHP and MEHP during its disposition in vivo. At present we can not choose between three possible explanations for the incorporation of 3H from dual-labeled DEHP into liver DNA. Tritium is found in normal DNA bases after exposure to 7-methylbenz[a] anthracene3H' [40] and the mechanism of incorporation is thought to involve an exchange the details of which are unknown. The binding of some fragment larger than C-1 from the ethylhexyl unit could conceivably be associated with a kinetic isotope effect that discriminates against 3H. Finally, the 3H may enter DNA through an independent route, involving a metabolite of DEHP that has lost its 1'-'4C. For example, 2- and 4-heptanones are produced by decarboxylation of 2-ethylhexanoic acid in rats [25], in amounts similar to that of the CO2 released. A choice between these alternatives, as well as a direct demonstration of the incorporation of C-1 from the ethylhexyl group of DEHP into C-2 of pyrimidine bases, must await the availability of DNA of much higher specific activity than 200 dpm/mg.

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