Chem. Res. Toxicol. 1989, 2, 260-266
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Metabolic Activation and Nucleic Acid Binding of Acetaminophen and Related Arylamine Substrates by the Respiratory Burst of Human Granulocytes Michael D. Corbett,*ptJ Bernadette R. Corbett,? M a r i e - H e l e n e Hannothiaux,? and Sergio J. Q u i n t a n a t Food Science and H u m a n Nutrition Department and Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32611 Received March 10, 1989
Following stimulation with phorbol myristate acetate, human granulocytes were found t o incorporate acetaminophen, p-phenetidine, p-aminophenol, and p-chloroaniline into cellular DNA and RNA. Phenacetin was not incorporated into nucleic acid or metabolized by such activated granulocytes. None of the substrates gave nucleic acid binding if the granulocyte cultures were not induced to undergo the respiratory burst. Additional studies on the binding of acetaminophen to DNA and RNA were made by use of both ring-14C-labeled and carbonyl-14C-labeled forms of this substrate. T h e finding t h a t equivalent amounts of these two labeled acetaminophen substrates were bound to cellular DNA demonstrated t h a t the intact acetaminophen molecule was incorporated into DNA. On the other hand, the finding t h a t excess ringJ4C-labeled acetaminophen was incorporated into cellular RNA implies partial hydrolysis of the acetaminophen substrate prior to RNA binding. Evidence was presented which strongly indicates that the nucleic acid binding of the substrates was covalent in nature. T h e inability of the respiratory burst to result in the binding of phenacetin to nucleic acid suggests t h a t arylamides are not normally activated or metabolized by activated granulocytes. Acetaminophen is a n exception to the recalcitrance of arylamides to such bioactivation processes because it also possesses the phenolic functional group, which, like the arylamine group, is oxidized by certain reactive oxygen species. Myeloperoxidase appears to be much more important in the binding of acetaminophen to DNA than it is in the DNA binding of arylamines in general. The role of the respiratory burst in causing the bioactivation of certain arylamines, which are not normally genotoxic via the more usual microsomal activation pathways, was extended to include certain amide substrates such as acetaminophen.
Introduction We recently reported on the covalent binding of a series of four arylamines to cellular nucleic acids as the result of the respiratory burst of human granulocytes ( I ) . A strong dependency was found between the electronic properties of the various arylamines and their relative ability to bind RNA and DNA. We proposed that the action of certain respiratory burst products on arylamines might cause the bioactivation of some members of this chemical group that are not susceptible to activation by the more classical processes associated with microsomal oxidases. In a continuation of studies designed to further elucidate the effect of chemical structure on the ability of arylamines and related chemicals to undergo respiratory burst dependent nucleic acid binding, we have investigated the arylamide/amine pairs of phenacetinlp-phenetidine and acetaminophenlp-aminophenol. Only phenacetin was found not to give any detectable nucleic acid binding. This was in agreement with our more general observation (unpublished results) that, unlike arylamines, arylamides do not bind to nucleic acids during the respiratory burst. An exception to this rule has been found in the case of acet*Address correspondence to this author at the University of Florida, IFAS-0163, Gainesville, FL 32611-0163. 'Food Science and Human Nutrition Department. Department of Pharmacology and Therapeutics.
*
0893-228x/89/2702-0260$01.50/0
aminophen. However, we were not surprised to observe nucleic acid binding by acetaminophen since it possesses the readily oxidizable phenol group. On the other hand, reports that acetaminophen does not give any nucleic acid binding as the result of the actions of either microsomes (2) or several peroxidative enzymes (3, 4 ) caused us to consider this pathway more closely. We now report on the results of our studies with these two arylamide/amine pairs, including this relatively unique ability of granulocytes to cause nucleic acid binding of acetaminophen.
Experimental Section Radiolabeled S u b s t r a t e s . [ring-UL-14C]Phenacetin and [ring-UL-14C]acetaminophen were obtained from Sigma Chemical Co (St. Louis, MO) and purified by chromatography on silica gel 60 (E. Merck, 70-230 mesh) columns (1.1X 1618 cm bed volume) by gravity elution with CH2C12-basedsolvents containing 0.5% and 2% methanol, respectively. HPLC on C18 reverse-phase columns with aqueous methanol solvents w&s employed to monitor the column effluents. Appropriate pure fractions were combined and diluted with the unlabeled compound (in the case of phenacetin), and then the specific activity of each pure compound was determined by HPLC and LSC; and the radiochemical purity was determined t o exceed 99.5%. [~arbonyl-l-'~C]Acetaminophen was prepared from [l-14C]aceticacid (Na+ salt; Chemsyn Science Laboratories, Lenexa, KS) after the conversion of the latter to [ l-'4C]acetic anhydride with acetyl chloride, followed by reaction with purified p-aminophenol. The [carb~nyl-l-'~C]acetinophen was purified in a manner similar to that described above for the purification of the ring-labeled analogue. [ring-UL-14C]-p-
0 1989 American Chemical Society
Chem. Res. Toxicol., Vol. 2, No. 4, 1989 261
Nucleic Acid Binding of Acetaminophen Aminophenol and [ring-UL-14C]-p-phenetidine were prepared by and the acid-catalyzed hydrolyses of [n'ng-UL-14C]acetamin~phen [ring-UL-*4C]phenacetin,respectively. The products were finally purified by gravity-column chromatographyon silica gel with 5% and 1% methanol in CHZCl2 for p-aminophenol and pphenetidine, respectively. Argon-purged solvents were used throughout the synthetic and purification procedures for these two compounds. HPLC was employed to monitor the purification of each substrate, and in conjunction with LSC to determine their specific activity. The radiochemical purities of both compounds were in excess of 98%. All 14C-labeledsubstrates were stored as 10 mM solutions in 95% ethanol under Argon and at -20 "C in the dark. The specific activities of the five substrates were as follows: [ring-UL-14C]phenacetin (9.6 mCi/mmol), [ring-UL14C]acetaminophen (7.5 mCi/mmol), [~arbonyl-l-'~C]acetaminophen (15.9 mCi/ mmol), [ring-UL-14C]-p-aminophenol (7.5 mCi/mmol), and [ring-UL-14C]-p-phenetidine (9.6 mCi/mmol). HPLC Methods. All HPLC analyses were conducted under isocratic conditions with a pBondapak C18column (3.9 mm X 30 cm, Waters Associates) at a flow rate of 1.5 mL/min. Peak detection was achieved by use of a Waters Model 440 UV detector with 254-, 280-, and/or 313-nmwindows. Solvent systems employed included the following: (a) 10% aqueous methanol, pH 7 (0.01 M KH2P04buffer), for analysis of acetaminophen ( t R = 8.0 min) and p-aminophenol ( t R = 3.5 min); (b) 40% aqueous methanol, pH 7 (0.01 M KHzP04buffer), for analysis of phenacetin ( t R = 6.8 min) and p-phenetidine ( t =~ 6.0 min); (c) methanol/HzO/HOAc/ethyl acetate (30:68:2:0.1)for attempted detection of the dimers and trimers of acetaminophen (5);and (d) methanol/H20/HOAc (10:89:1) containing 5 mM heptanesulfonic acid for attempted detection of the glutathione conjugates of acetaminophen (6). Incubation of Substrates with Granulocyte Suspensions.
The granulocyte fraction was isolated from freshly collected human blood as previously described ( I ) . The incubations were conducted as previously described (I),generally employing 30-mL volumes containing 2 X lo6 cells/mL. The substrates as 10 mM solutions in 95% ethanol were added to give a concentration of 10 pM, except where otherwise noted, followed by PMA to initiate the respiratory burst. Inhibitor studies with NaN3,catalase, and superoxide dismutase were carried out by preincubation of the cell culture with the inhibitors for 5 min prior to the addition of PMA. The occurrence of a normal respiratory burst was monitored by the cytochrome c reduction method (7) for superoxide anion production. The incubation was generally terminated after 30 min by rapidly chilling the cell suspension, followed by centrifugation at 600g for 10 min at 4 "C. The supernatant was decanted from the cell pellet and analyzed by HPLC methods for the amount of substrate remaining and, in certain cases, for the presence of suspected metabolites. Isolation of DNA and RNA from Granulocyte Pellets. The previously described method ( I ) was slightly modified in two places. The lysis of the cells was facilitated through the use of a Potter-Elvehjem tube, which reduced the totallysis time to only 1-1.5 h. Following the treatments of DNA and RNA with RNase and DNase, respectively, the proteinase K treatment step was eliminated and immediate extraction with phenol reagent was employed. Studies on the Nature of Substrate Binding to DNA and RNA. Dialyses of portions of combined DNA from multiple
incubations with the substrates p-phenetidine and both the ringand carbonyl-labeled acetaminophen were conducted according to the method described by Andersson et al. (8). Enzymatic hydrolysis of DNA and solvent partitioning studies were carried out in a manner similar to that described previously (1). Hydrolysis of RNA was accomplished in a similar manner by the substitution of RNase T Iand RNase A for DNase. For all hydrolysates, single extractions with H20-saturated diethyl ether and 1-butanol were made, followed by two sequential extractions with HzO-saturated 1-butanol containing 100 mM tetrabutylammonium phosphate.
Results Slight modifications were made in the original method (I) to isolate DNA and RNA from granulocytes, since it
Table I. Binding of ["CIAryiamine Substrates to Granulocyte DNA and RNA nmol of nmol of
substrate" phenacetin
[14C]arylamine/ [14C]arylamine/ re1 binding, mg of DNAb mg of RNAb RNA/DNA
