Identification of the Major Protein Adduct Formed in Rat Liver After Thioacetamide Administration1

[CANCER RESEARCH 41. 3430-3435, September 1981] 0008-5472/81 70041-0000$02.00 Identification of the Major Protein Adduct Formed in Rat Liver after Thioacetamide Administration1 Martin C. Dyroff2 and Robert A. Neal3 Center in Environmental Toxicology, Department of Biochemistry. Vanderbilt University School of Medicine. Nashville, Tennessee 37232 ABSTRACT that metabolism of thioacetamide-S-oxide by rat liver micro- somes results in the formation of a reactive metabolite(s) which The in vivo covalent binding of the hepatocarcinogen thioa- binds to calf thymus DNA, calf liver RNA, polyguanylate, and cetamide to rat liver protein has been examined. Following administration of 3H- or '"C-labeled thioacetamide, the modified polyadenylate (23). In the present investigation, we have de termined the structure of the major amino acid adduct of liver amino acids present in the hepatic cytosolic proteins were proteins formed on administration of [3H]- or [1-'"C]thioaceta- isolated by enzymatic digestion and ion-exchange chromatog- mide to rats. raphy. Approximately 70% of the radioactivity covalently bound to cytosolic protein was recovered in a compound which upon MATERIALS AND METHODS acid hydrolysis yielded lysine and radiolabeled acetate. Addi tional studies indicated the structure of this adduct was N-e- Chemicals. [3H]Thioacetamide (specific activity, 25 mCi/mmol) and acetyllysine. [1-'4C]thioacetamide (specific activity, 1 mCi/mmol) were synthesized from [3H]acetonitrile (3.75 Ci/mmol) and [1-'4C]acetonitrile (10 mCi/ mmol) as described previously (17, 25). The [3H]acetonitrile was the INTRODUCTION product of New England Nuclear, Boston, Mass., and the [1-'"C]ace- Thioacetamide produces centrilobular hepatic necrosis (1, tonitrile was obtained from ICN Chemical Radioisotopes Div., Irvine, 8), bile duct proliferation (4, 6), and liver cirrhosis (4, 7) in rats. Calif. The purity of the radiolabeled thioacetamide was determined by thin-layer chromatography (17) to be greater than 98%. [D3]Thioacet- Thioacetamide is rapidly metabolized in vivo in the rat to sulfate (15), acetamide (20), and thioacetamide-S-oxide (2,16). Acet- amide was synthesized using [D3]acetonitrile (Pfaltz and Bauer, Inc., Stamford, Conn.) as described previously (25). PhÃ©nobarbital, 3- amide does not produce hepatotoxic effects such as those methylcholanthrene, pronase, leucine amino peptidase, and amino acid observed with thioacetamide even at very high doses (3). standards were obtained from Sigma Chemical Co., St. Louis, Mo.; W- However, an equimolar dose of thioacetamide-S-oxide pro e-acetyllysine was obtained from Vega-Fox Biochemicals, Tucson, duces a more severe necrosis and at an earlier time after Ariz.; dimethylformamide dimethylacetal and 3% OV-17 on Chromo- administration than does thioacetamide (8, 16). Following ad sorb W(HP) were from Pierce Chemical Co., Rockford, III.; Porapak Q ministration of [3H]- (16, 20) or [1-14C]thioacetamide (16), co was from Alltech Associates, Arlington Heights, III.; Sephacryl S-200 valent binding of radioactivity to cellular macromolecules can was obtained from Pharmacia Fine Chemicals, Inc., Piscataway, N. J.; be detected. After administration of [35S]thioacetamide or Dowex 50W-X8 was from Bio-Rad Laboratories, Richmond, Calif.; and [35S]thioacetamide-S-oxide to rats, only trace amounts of co the 4.6-mm x 25-cm Zorbax octadecyl silane column was the product of DuPont, Wilmington, Del. Omnifluor was obtained from New England valently bound radioactivity (16) can be detected. However, Nuclear, and Nuclear-Chicago solubilizer and ACS were from Amer- Raw and Rockwell (19) have reported a significantly higher sham/Searle Corp., Arlington Heights, III. labeling of the protein in the nucleolus as compared to other Animals. Male Sprague-Dawley rats (HarÃanIndustries) (150 to 160 subcellular fractions in rabbits 2 hr after administration of g) were fed a commercial diet of Purina rat chow ad libitum. The [35S]thioacetamide. cytochrome P-450 monooxygenase activity of rat liver was induced by Rees ef al. (20) proposed that the incorporation of radioac i.p. injection of phÃ©nobarbital (80 mg/kg) in 1.15% KCI at 72, 48, and tivity from [3H]thioacetamide into macromolecules was the re 24 hr or 3-methylcholanthrene (40 mg/kg) in corn oil 48 hr prior to administration of [3H]thioacetamide (2.67 mmol/kg; 2 Â¿iCi/g body sult of conversion of the metabolite acetamide to acetate which weight), [1-14C]thioacetamide (2.67 mmol/kg; 0.33 Â¿iCi/g body entered the metabolic pool and became incorporated into weight), or [D3]thioacetamide (2.67 mmol/kg). Following thioacetamide endogenous molecules. However, Porter ef al. (16) have dem administration, the rats were fasted but allowed unlimited access to onstrated that administration of a dose of radiolabeled aceta water until sacrifice 12 hr later. mide, estimated to approximate the levels formed in vivo after Subcellular Fractionation. Rats were stunned by a blow to the head thioacetamide administration, does not result in significant and decapitated. The livers were perfused in situ with ice-cold 1.15% radiolabeling of hepatic macromolecules. KCI prior to excision and immersion in ice-cold 1.15% KCI. A portion Recent results have suggested that the reactive metabolite of each liver was removed for determination of its content of radioac of thioacetamide which becomes covalently bound is a further tivity by combustion in a Packard Tri-Carb tissue oxidizer. Subcellular oxidation product of thioacetamide-S-oxide, perhaps thioacet- fractionation of the remainder of each liver was performed as described amide-S-dioxide (8, 17), formed in a reaction catalyzed by previously (5). Purified nuclei were prepared by the hypertonic sucrose amine oxidase (18, 26), the cytochrome P-450-containing method of Spelsberg ef al. (21). All subcellular fractions obtained by centrifugation were resuspended in the homogenization buffer to a final monooxygenases (8, 16), or both. It has also been reported concentration of 2 g wet weight liver per ml. Aliquots of each subcellular 1This work was supported by Grants ES 00075 and ES 00267. fraction were analyzed for protein by the method of Lowry ef al. (11 ), 2Recipient of Grant ES 07028. using bovine serum albumin as the standard, and for radioactivity, by 3To whom requests for reprints should be addressed. liquid scintillation counting after solubilization with Nuclear-Chicago Received March 20, 1981; accepted June 11,1981. tissue solubilizer and neutralization with glacial acetic acid. 3430 CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1981 American Association for Cancer Research. Hepatic Protein Adduci of Thioacetamide Determination of Covalent Binding. Protein and other macromole- Amino Acid. The radioactive compound recovered from the amino acid cules present in aliquots of the subcellular fractions corresponding to analyzer and purified from buffer components was derivatized using 1 g, wet weight, of liver were precipitated by adding 20 volumes of dimethylformamide dimethylacetal as described by Thenot and Horning cold 95% ethanol. The precipitated material was homogenized in the (22). The derivatized compound was chromatographed on a 2-mm x 6- 20 volumes of ethanol using a Son/all Omnimixer at top speed for 30 ft column of 3% OV-1 7 on Chromosorb W(HP). The Chromatographie sec and then centrifuged for 10 min at 20,000 x g. This cycle was conditions were: flame ionization detector, 275Â°; injector, 250Â°; and repeated until the radioactivity in the supernatant was less than 2 times column, 200Â° for the first 10 min and then temperature programmed background (5). The radioactivity associated with the remaining pre 15Â°/min to a final temperature of 250Â°. The carrier gas was helium cipitated material was considered to be covalently bound. This material maintained at a flow rate of 20 ml/min. Alternatively, the 3H-labeled was solubilized by heating for 1 hr at 60Â°in N NaOH and was assayed adduct was initially esterified with methanol:N HCI at 100Â° for 15 min. for protein and radioactivity. After removal of the solvent, the resulting mixture was reacted with Aliquots of the cytosolic fraction were chromatographed on a 1.5- x trifluoroacetic anhydride in mÃ©thylÃ¨nechloride at room temperature for 50-cm column of Sephacryl S-200. The column was eluted with 0.05 15 min. The solvent was removed under a stream of nitrogen, and the M sodium phosphate buffer, pH 7.4, and the effluent was monitored for sample was dissolved in methanol and analyzed on a 2-mm x 6-ft UV absorbance at 280 nm and for radioactivity. Aliquots of the hepatic column of 3% OV-17 in the gas Chromatograph. The Chromatographie cytosolic fraction were also analyzed by sodium dodecyl sulfate:poly- conditions were: flame ionization or electron capture detector, 260Â°; acrylamide gel electrophoresis on 7.5% gels according to the method injector, 210Â°; and column, 140Â° initially and then programmed 10Â°/ of Laemmli ef al. (9). The gels were stained with Coomassie Brilliant min to a final temperature of 250Â°. The carrier gas was nitrogen and Blue and scanned for absorbance at 595 nm. Subsequently, the gels maintained at a flow rate of 20 ml/min. Radioactivity was monitored by were sliced in 2-mm pieces, the slices were solubilized using 30% bubbling the column effluent through 18 ml of ACS scintillation cocktail H2C>2and 70% HCICX,according to the method of Mahin and Lofberg for timed intervals and counting the timed fractions. (12), and analyzed for radioactivity. A Finnigan Model 3200 GC-MS, operated in the electron impact Enzymatic Hydrolysis of Hepatic Cytosolic Protein. Cytosolic frac mode, was used to obtain the mass spectrum of the thioacetamide- tions from the livers of rats administered [3H> or [1-14C]thioacetamide modified amino acid which was derivatized using the procedure de were dialyzed for 24 hr at 4Â°against 0.05 M phosphate buffer, pH 7.4, scribed by Thenot and Horning (22).

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