comparative adduct formation of 4-aminobiphenyl and 2 ... · an initial n-oxidation (7), followed...

[CANCER RESEARCH 36, 2374-2381, July 1976]

Comparative Adduct Formation of 4-Aminobiphenyl and 2-Aminofluorene Derivatives with Macromolecules of Isolated Liver Parenchymal Cells

Charles M. King, 2 Nancy Raab Traub, Raymond A. Cardona, 3 and Roger B. Howard

Division of Cancer Research, Department of Medicine, Michael Reese Hospital and Medical Center [C. M. K., N. R. T., R. A. C., R. B. H.], Chicago 60616, and the University of Chicago Pritzker School of Medicine [C. M. K.], Chicago, Illinois 60637

SUMMARY

Isolated parenchymal cells of rat liver have been used in a study of the metabol ic activation of derivatives of the carcinogens 4-aminobiphenyl and 2-aminof luorene. The formation of adducts of these compounds with cellular RNA and protein has been taken as evidence of their transformation to metabol i tes that are capable of spontaneous reac- t ion with tissue macromolecules.

The hydroxamic acid N-hydroxy-N-4-acetylaminobiphenyl was bound to RNA to a greater extent than were the amino-, hydroxylaminoo, nitroso-, nitro-, acetylamino-, or azoxybiphenyl derivatives. RNA adducts of the hydroxamic acid retained litt le of the acetyl group. The structural requirements for b inding and the nature of the bound derivatives are consistent with the activation of N-hydroxy-N-4-acetylaminobiphenyl by N --, O acyltransfer. Approximately equal quantit ies of 4-nitrosobiphenyl and the hydroxamic acid were bound to protein, but far less of the nitroso derivative was incorporated into RNA.

Adduct format ion of N-hydroxy-N-2-acetylaminof luorene with RNA occurred with retention of the acetyl group and was dependent on the concentrat ion of sulfate in the media. Consequently, reaction of the f luorenyl derivative with RNA probably resulted from conjugat ion of the hydroxamic acid with sulfate.

INTRODUCTION

Events crucial to the init iation of carcinogenesis by chemicals are believed to involve the alteration of tissue macromolecules. Derivatives of the carcinogenic arylamines AAF 4

These studies were supported by a grant from the Jules J. Reingold Trust, by USPHS Grants CA 13179 from the National Cancer Institute and CA 15640 from the National Cancer Institute through the National Bladder Can- cer Project, and by the Medical Research Institute Council of Michael Reese Hospital and Medical Center.

2 Present address: National Center for Toxicological Research, Jefferson, Ark. 72079.

3 Present address: Uniroyal Chemical, Agricultural Chemicals Research, Elm Street, Naugatuck, Conn. 06770.

4 The abbreviations used are as follows. Alternate names are given in parentheses. Abbreviations for unlabeled compounds are given in the text without isotope designation. AAF, 2-acetylaminofluorene; [3H]ABP, 4-amino- [3H]biphenyl; AF, 2-aminofluorene; N-hydroxy-[l"C,3H]AAF, N-hydroxy-N- [2'-3H]acetyl-2-amino[9J4C]fluorene (N-2-fluorenylacetohydroxamic acid); N-hydroxy-[l"C,3H]AABP, N-hydroxy-N-[carbonyl- 14C]acetyl-4-amino[3H]biphenyl (N-4-biphenylacetohydroxamic acid); nitro-[3H]BP, 4-nitro[3H]bi -

and ABP are known to bind covalently to nucleic acid and protein fo l lowing metabolic activation in v ivo (35) or in v i t ro

(2,3, 8, 9, 20, 25, 26). These reactions are thought to involve an initial N-oxidat ion (7), fol lowed by secondary activation steps (37, 44). The induct ion of tumors in various tissues of several species may relate, in part, to the distr ibut ion of enzymes that are capable of activating these N-oxidized metabolites (2, 8, 21,24).

Metabolism studies in v ivo of excretory products or covalently bound derivatives yield results that reflect systemic transformations of the chemical and reflect on ly indirectly the actual activation processes. Products that are stable enough to be excreted are unlikely to be reactive species, and derivatives bound covalently to tissue macromolecules cannot be related definit ively to the enzyme systems responsible for their formation.

Metabolic activation studies with subcel lu lar fractions have relied primari ly on the reaction of intermediates with exogenous trapping agents (9, 22, 25, 26). A l though these experiments have facil i tated the partial characterization of these enzyme systems, they do not take into account the metabolic controls that exist within the intact cell.

The use of isolated intact cells should c i rcumvent some of these diff icult ies by permitt ing comparative studies of the activation of dif ferent derivatives in a system that is subject to the cellular controls and metabolic capabil i t ies of the tissue wi thout addit ional systemic t ransformat ion. In this study, we have examined the reaction of a number of metabolic derivatives of ABP and AF with RNA and protein of isolated rat liver parenchymal cells. The variat ion in binding levels of these derivatives within a system where compound concentrat ion, cofactors, and cell numbers are control led indicates the relative contr ibut ion of these compounds to the alteration of tissue macromolecules in intact cells and contr ibutes to our understanding of the activation processes that must occur prior to the actual b ind ing events.

MATERIALS AND METHODS

Chemicals. The fol lowing were obtained f rom the com- mercial sources indicated: di thiothrei tol , Pronase (nuclease

phenyl; [14C,3H}AABP, N-[carbonyl-l"C]acetyl-4-amino[3H]biphenyl (N-4-bi- phenylacetamide); N- hyd roxy-[3H]ABP, N- hydroxy- 4- amino[3H]biphenyl; nitroso-[3H]BP, 4-nitroso[3H]biphenyl; azoxy-[3H]BP, 4,4'-azoxy[3H]biphenyl. aminobiphenyl; N-hydroxy-AAF, N-hydroxy-N-acetyl-2-aminofluorene; nitro- BP, 4-nitrobiphenyl; azoxy-BP, 4,4'-azoxybiphenyl.

Received August 8, 1975; accepted April 9, 1976.

2374 CANCER RESEARCH VOL. 36

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free), and yeast tRNA (Calbiochem, Los Angeles, Calif.); sucrose (special enzyme grade, Schwarz/Mann, Orange- burg, N. Y.); RNase A and collagenase (Type I) (Worthington Biochemical Co., Freehold, N. J.).

N-hydroxy-[14C,3H]AAF (6.1 mCi 14C, and 18.7 mCi 3H per mmole), N-hydroxy-[14C,3H]AABP (11.9 mCi ~4C and 30.7 mCi 3H per mmole), N-hydroxy-[3H]AABP (281 mCi/mmole), and nitro-[3H]BP (231 mCi/mmole) were prepared and the radiochemical purity established as described previously (6).

[3H]AABP (158 mCi/mmole) was obtained by the reduction of nitro-[3H]BP to [3H]ABP with hydrazine and 10% palladium on charcoal (4), followed by acetylation of the amine in benzene with acetic anhydride (38). [~4C]AABP was prepared in 71% yield by the reaction of equimolar quantities of ABP, [1J4C]acetyl chloride (12.6 mCi/mmole) (California Bionuclear Corp., Sun Valley, Calif.), and trieth- ylamine in ether. Double-labeled ['4C,3H]AABP was prepared to give a final specific activity of 8.6 mCi 14C per mmole and 36.8 mCi 3H per mmole. Radiochemical purity was established by chromatography on thin layers of silica gel (Eastman No. 13181, Eastman Kodak Co., Rochester, N. Y.) using diethyl ether:hexane (7:3) as solvent. Analysis of the chromatograms by elution with a toluene-based scintillation solution, or by radioautography, revealed a single radioactive component that cochromatographed with authentic unlabeled AABP (RF 0.35).

[3H]ABP (200 mCi/mmole) was prepared in 44% yield by hydrolysis of [3H]AABP in ethanolic HCI (22). The amine derivative cochromatographed on thin layers of silica gel with authentic ABP as a single radioactive component (RF 0.4) using hexane:diethyl ether (1:1) as solvent.

N-Hydroxy-[3H]ABP (231 mCi/mmole) was prepared in 79% yield by the reduction of nitro-[3H]BP with ammonia and hydrogen sulfide in a solution of equal parts 95% ethanol and dimethylformamide (42). The hydroxylamine was re- crystallized from CHCI3 by reducing the volume of the solution with a stream of N2. Greater than 90% of the trit ium migrated with authentic N-hydroxy-ABP (RF 0.46) on silica gel using benzene:ethanol (98:2) as solvent. The balance of the radioactivity consisted of approximately equal quantities of azoxy-[3H]BP (RF 0.9) and [3H]ABP (RF 0.76).

Nitroso-[3H]BP (196 mCi/mmole) was obtained in 29% yield by oxidation of the N-hydroxy-[3H]ABP with ferric am- monium sulfate in 0.4 M sulfuric acid in dimethylformamide (1). Thin-layer chromatography on silica gel using benzene:hexane (1:1) as solvent indicated that approximately 90% of the label was nitroso-[3H]BP (RF 0.63), with the major contaminant (6%) being azoxy-[3H]BP (RF 0.47).

Azoxy-[3H]BP (307 mCi/mmole) was recovered as a by- product of the nitroso-[3H]BP synthesis. The CHCI3-soluble, hexane-insoluble, yellow material was shown to be azoxy-BP by comparison on thin-layer chromatography to unlabeled azoxy-BP that had been characterized by UV spectroscopy, melting point (43), and chromatography on silica gel as described above.

Animals. Adult male Sprague-Dawley-derived rats were raised and maintained in our animal facilities at 24 ~ in stain- less steel cages with solid floors covered with soft-wood

Activation of Arylamines by Liver Cells

shavings. Distilled water and Purina laboratory chow (Ral- ston Purina Co., St. Louis, Mo.) were available continu- ously.

Incubation of Isolated Parenchymal Cells with Arylam- ine Derivatives. Isolated liver parenchymal cells were obtained by use of an enzymatic dispersion technique (13). Livers were perfused with collagenase in calcium-free Eagle's minimum essential medium based on Hanks' solution. Calcium was then added during the subsequent incubation of liver slices with collagenase, as previously described (13). Cell counts were measured with a hemocyto- meter. Viability was greater than 95%, as determined by trypan blue exclusion. Approximately 3 • 106 cells were suspended in 2 ml of Hanks' solution in a 25-ml sil iconized Erlenmeyer flask. All incubations were carried out in duplicate. Labeled compounds were dissolved in dimethyl sulf- oxide, 10/~1, for addition to the cells. Cell preparations were incubated with shaking at 37 ~ in air. The incubations were terminated and the cells were collected by low-speed centrifugation at 4 ~ followed by homogenization with a glass homogenizing tube and pestle in 2 ml Hanks' solution and 2 ml buffer-saturated phenol. MgCI2 replaced the MgSO4 in sulfate-free incubations; acetate was added as the sodium salt where indicated.

Isolation of RNA. The homogenate was extracted twice at 4 ~ with buffer-equilibrated phenol; the 1st extraction was for 1 hr and the 2nd was for 15 min. Two volumes of ethanol:m- cresol (9:1, by volume) were added to the aqueous phase, precipitating total RNA overnight at 4 ~ The RNA precipitate was collected by centrifugation, washed successively with 70 and 95% ethanol, and dried over CaSO4 under vacuum. The RNA was dissolved in 0.015 M NaCI:0.0015 M sodium citrate, pH 7.0, and the absorbance at 260 nm was determined. The isotopic contents of 0.1-ml aliquots in 10 ml scintillation fluid (3a70B, Research Products International, Elk Grove, II1.) were determined in a scintil lation counter (Isocap-300, Searle Analytic, Des Plaines, III.) using an auto- matic external standardization technique to determine counting efficiency. Activity was expressed as nmoles bound per mg RNA, assuming that 20 A2~,, units are equiva- lent to 1 mg of RNA. Usually, 1 to 3 A2,~ units of RNA were recovered from each flask of 3 x 106 cells.

Isolation of Protein. Protein was precipitated from the phenol layer of the cell homogenate by addition of an excess of methanol:ether (1:2) (41). The precipitate was collected by low-speed centrifugation; washed successively with methanol:ether (2 times), 95% ethanol (3 times), and ether (2 times); and dried under vacuum. Each flask of 3 x 10 e cells usually yielded 6 to 8 mg of protein.

Aryl nitroso derivatives can react nonenzymatically with protein (32). Consequently, in experiments with nitroso-BP and N-hydroxy-ABP, cells were washed 3 times with 95% ethanol prior to homogenization in order to prevent pro- longed exposure of the proteins to nitroso derivatives in the phenol layer.

Protein samples were weighed on a microbalance, moist- ened with 0.1 ml 0.2 M NaOH, and incubated at 37 ~ with 1 ml solubilizer (NCS, Amersham Searle, Arl ington Heights, III.). Following the addition of a toluene-based scintil lation fluid,

JULY 1976 2375



C. M. King et al.

3H and 14C were determined as previously described (21). Activity was expressed as nmoles of substrate bound per mg protein.

Arylhydroxamic Acid Acyltransferase Assay. Dispersed liver parenchymal cells were homogenized in 0.05 M PPi:NaCI:I mM dithiothreitol, pH 7.0. Cytosol was prepared by centrifugation of the homogenate at 105,000 x g (aver- age) for 1 hr at 4 ~ The ability of the 105,000 x g supernatant to introduce the AF or ABP moiety of the hydroxamic acids into tRNA by N ---, O acyltransfer was determined as previously described (21). Duplicate assays at several concentrations of soluble fraction were incubated for 20 min with 1 mg tRNA. Activity was expressed as nmoles substrate bound to nucleic acid per mg protein in the soluble fraction. Protein was determined by a modified Folin method (34), with bovine serum albumin as a standard.

Sulfotransferase Assay. The previously reported method for assay of the sulfate-dependent incorporation of acetyl- labeled fluorene or biphenyl hydroxamic acids into tRNA was used (24). Liver cells were homogenized in 0.1 M Tris:HCI buffer, pH 7.4. Cytosols, prepared as described above, were assayed in duplicate by incubation with ATP, Na2SO,, MgCI2, and 3 mg tRNA in volume of 1 ml for 1 hr (24). Activity was expressed as nmoles substrate bound to tRNA per mg protein.

Sucrose Density Gradient Centrifugation of RNA. Prepa- rations of RNA were subjected to centrifugation on 16 ml 5 to 20% sucrose gradients in 0.1 M NaCI:0.01 M sodium acetate, pH 5 (17). Centrifugation was for 18 hr at 24,000 rpm, 4 ~ , in a SW 27.1 rotor (Spinco Instruments, Palo Alto, Calif.). The gradients were analyzed by use of a UV monitor and fract ionating device (Isco Instruments Co., Lincoln, Nebr.). Fractions (1 ml) were diluted with 10 ml of 3a70B scinti l lation f luid and then counted in a liquid scinti l lation counter as described above.

RESULTS

Activation of Arylhydroxamic Acids by Enzymes of Is- olated Liver Parenchymal Cells. Prior to the use of isolated liver cells for comparative studies on the metabolism of arylamine derivatives, it seemed essential to determine whether the cells retained the soluble liver enzymes that had previously been shown to catalyze the formation of nucleic acid adducts in vitro. Assay of 105,000 x g superna- tants of liver cells for acyltransferase and sulfotransferase using N-hydroxy-[14C,3H]AAF or N-hydroxy-[14C,3H]AABP as substrate demonstrated that these enzymes were active constituents of the isolated cells (Table 1). Acyltransferase- induced adduct formation was twice as great when N-hydroxy-[14C,3H]AAF rather than N-hydroxy-[14C,3H]AABP was the substrate.

Isolation of RNA from Incubated Liver Cells. The cells remained approximately 90% viable on incubation with labeled arylamine derivatives for periods of up to 2 hr. RNA recovered from these incubations, usually 1 to 3 A2,0 units, had an absorbance maximum at 258 nm and A2,o/A28n of approximately 2. Analysis of sucrose density gradients obtained by centr i fugation of the RNA disclosed 3 UV-absorb-

ing peaks that were consistent with those of the 28, 18, and 4 to 5 S species of rRNA and tRNA. These peaks were coincident with the radiolabel present in the gradients. A typical profile is shown in Chart 1. Treatment of RNA with RNase for 30 min produced an altered profile with all radioactivity and UV-absorbing material appearing at the top of the gradient. Treatment with Pronase did not alter the UV or radiochemical profiles of the gradients. When labeled RNA was treated with RNase, and carrier RNA was added to the solution after incubation, 95% of the radioactivity was no longer precipitable on addition of 2 volumes of 2% potassium acetate in 95% ethanol. Similar incubations with Pro- nase yielded no more ethanol-soluble radioactivity than did control incubations that contained no enzyme.

Combination of Biphenyl Derivatives with RNA and Pro- tein of Liver Cells. Experiments in which liver cells were incubated with N-hydroxy-[3H]AABP for up to 4 hr demonstrated that the amounts of label bound to protein and RNA were related to the length of the incubation period (Chart 2). The rate of incorpoi;a.tion was greatly reduced after 2 hr of incubation. Incorporation into RNA was minimal in control experiments in which medium was quickly removed and the cells were homogenized in phenol. Significant quantities of the label were bound to the proteins in the interval required for removal of the medium and homogenization of the cells. In further experiments with N-hydroxy-['4C,3H]AABP, it was

Table 1 Activation of N-hydroxy-AABP and N-hydroxy-AAF by N - , 0

acyltransferase and sulfotransferase of isolated parenchymal liver cells

Cytosols of isolated liver cell homogenates were assayed for acyltransferase and sulfotransferase as described in "Materials and Methods." These values are the mean +_ S.D. from assay of cells from 3 separate preparations.

nmoles arylamine bound to RNA/mg protein

Substrate Acyltransferase Sulfotransferase

N-Hydroxy-AAF 1.3 _+ 0.2 45.0 _+_ 11.0 N-Hydroxy-AABP 0.6 -+ 0.2 3.0 _+ 1.1

o lj �9 7 0 0

CL

o ~

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--008. . 3 5 0 "<

u Q_

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Chart 1. Sucrose density gradient centrifugation of NNA isolated from liver cells treated with N-hydroxy-[3HlAABP. A 0.4-ml sample of RNA, containing 0.98 A=~ unit and 2330 dpm 3H, w a s cen tr i fuged and analyzed a s described in "Materials and Methods."




Activation of Arylamines by Liver Cells

shown that combinat ion of the hydroxamic acid occurred with loss of the acetyl group This observat ion indicated that adducts of protein were formed as a consequence of metabolism of the compound rather than as artifacts of the puri f icat ion techniques. The specif ic activity of the RNA, expressed as nmoles biphenyl bound per mg, was 65% that

E

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Time (hours/

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Chart 2. Influence of incubation time on the incorporation of N-hydroxy- [~H]AABP. Isolated liver cells (3.5 x 10 �9 cells per flask) were incubated with 20/~M N-hydroxy-[3H]AABP for 1-, 2-, or 4-hr intervals. RNA and protein were isolated as described in "Materials and Methods." The incorporation, in nmoles bound per mg, is shown on the ordinate. �9 protein adducts; O, RNA adducts.

of protein under condi t ions that were opt imal for format ion of nucleic acid adducts. The total amount of compound bound to protein and RNA was approx imate ly 14% of the N- hydroxy-AABP incubated.

The relative binding of biphenyl metabol i tes to RNA and protein of the liver cells was establ ished by compar ing the amount of adduct format ion of the metabol i te to that of N- hydroxy-[14C,3H]AABP. With a constant concentrat ion of compound, an inverse relat ionship existed between the number of cells incubated and the speci f ic activity of the RNA and protein isolated. Furthermore, di f ferent cell preparations exhibi ted varying capacit ies for the act ivat ion of N- hydroxy-[14C,3H]AABP. To contro l these interexper iment variat ions, dupl icate flasks conta in ing N-hydroxy- [14C,3H]AABP were included to serve as posit ive contro ls in exper iments with each liver cell preparat ion and to serve as a reference compound in determin ing the relative b inding of the various bound derivatives.

Chart 3 presents data that descr ibe the concent ra t ion dependence of the combinat ion of the ABP derivatives with RNA and protein. Combinat ion of each biphenyl derivative with protein was always greater than with RNA. In general, higher concent ra t ions of labeled compounds were more effective in promot ing react ions with protein than with RNA. Incorporat ion of the acetyl group of AABP or N-hydroxy- AABP into protein was less than 14% that of the biphenyl incorporat ion; no label from the acetyl group of either of

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C o n c e n t r a t i o n ( . M ) Chart 3. Adduct formation of biphenyl derivatives with RNA or protein. Isolated liver cells were incubated with 3 concentrations of eaoh biphenyl derivative

for 2 hr. A standard concentration of N-hydroxy-[14C, 3H]AABP was included as a positive control and reference point. Control flasks in which the cells were immediately centrifuged after exposure to the highest concentrations of compound were included for each derivative. �9 0, [3H]biphenyl bound to protein; �9 - - - �9 ['4C]acetyl bound to protein; �9 [3H]biphenyl bound to RNA; II, N-hydroxy-[3H]AABP bound to protein; Iq, N-hydroxy-[3H]AABP bound to RNA; &, protein isolated from control flask; /k, RNA isolated from control flask.

JULY 1976 2377



C. M. King et al.

these compounds was detectable in the RNA recovered f rom this series of experiments. Control flasks in which the exposure of the cells to the highest concentrat ion of compound was held to the shortest period possible have been included in each series of experiments. Al though levels of binding to RNA in these controls were uni formly low, binding to protein varied considerably. Control experiments with nitroso-[3H]BP had 45% as much binding to protein as in those flasks that had been incubated for 2 hr, despite the extraction of cells with ethanol pr ior to homogenizat ion. This procedure substantial ly reduced the combinat ion of nitroso-[3H]BP and N-hydroxy-[3H]ABP with protein in these controls.

The hydroxamic acid was bound to RNA to a greater extent than was any other derivative tested (Table 2). Only about 50% as much [14C,3H]AABP or nitroso-[3H]BP and 25 to 30% as much [3H]ABP and N-hydroxy-[3H]ABP were bound to RNA. Nitro-[3H]BP and azoxy-[3H]BP, which is present .as a contaminant in the N-hydroxy-[3H]ABP and nitroso-[3H]BP preparations, did not react with RNA of the cells. Protein adduct format ion was most pronounced with the hydroxamic acid and nitros�9 derivatives, whi le the other derivatives were less reactive.

Influence of Sulfate and Acetate on the Metabolic Acti- vation of Arylamines by Liver Cells. Deacetylated arylamine residues of N-hydroxy-AABP consti tute the predominant form of the derivatives bound to RNA of liver cells in vitro as reported here, and in vivo as publ ished previously (30). In contrast, most of the f luorenyl residues of N-hydroxy-AAF that are attached to the RNA of the liver of male rats retain their acetyl group (17, 18, 28, 29) and are thought to result f rom activation of the hydroxamic acid by conjugat ion with sulfate (8, 9, 11,25, 26). Impl icat ion of sulfate conjugat ion in the activation process has come in part from comparative experiments in which some animals were depleted of sulfate by the administrat ion of large amounts of xenobiot ic compounds that are excreted as sulfate conjugates, and others were given large quantit ies of sulfate with these agents (10). The use of liver cells permitted study of the role of sulfate in the activation process by altering the sulfate concentrat ion of the media, rather than by introducing an- other organic compound that might interfere with the metabol ism of the aromatic amine by mechanisms not involv- ing sulfate conjugat ion.

Incubation of N-hydroxy-[14C,3H]AAF with liver cells in Hanks' solution that contained 0.8 mM sulfate disclosed that most of the f luorene nucleus was bound to the RNA with retention of the acetyl group (Chart 4). When sulfate was omitted form the media, combinat ion of acetylated derivatives of N-hydroxy-[14C,3H]AAF to RNA and protein was reduced by 90%. Comparison of the incorporat ion of the ring and acetyl labels disclosed that binding of deacetylated derivatives was essentially unchanged (Chart 5). Addit ional experiments with sulfate concentrat ions 10 times greater than that of Hanks' solut ion were no more effective than the standard solut ion in promot ing adduct format ion. Simi lar experiments with N-hydroxy-[14C,3H]AABP demonstrated that sulfate concentrat ion did not substantial ly affect incorporation of either the acetyl or biphenyl moieties into RNA or protein (Chart 5).

Table 2 Relative incorporation of biphenylamine derivatives into RNA and

protein of isolated rat liver cells These values are based on the data reported in Chart 3. For

comparative purposes, the relative abilities of these compounds to form adducts with protein or RNA in liver cells were established by assigning N-hydroxy-AABP a value of 100 and expressing the relative adduct formation of the other compounds as a percentage of the activity of the hydroxamic acid.

Relative binding

Compound RNA Protein

N-Hyd roxy-AABP 100 100 AABP 55 55 ABP 30 57 N-Hydroxy-ABP 28 37 Nitroso-BP 50 102 Nitro-BP 1 12 Azoxy-BP 4 15

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Chart 4. Incorporation of N-hydroxy-AAF into RNA and protein of isolated rat liver cells. Liver cells were incubated with 3 concentrations of N-hydroxy- ["C,SH]AAF for 2 hr as reported in "Materials and Methods." �9 �9 ['4C]- fluorenyl bound to protein; � 9 1 4 9 [3H]acetyl bound to protein; �9 0, ['4C]fluorenyl bound to RNA; 0- - -�9 [3H]acetyl bound to RNA; A, protein isolated from control flask; Z~, RNA isolated from control flask.

Other mechanisms by which biphenyl derivatives may be bound to RNA include O-esterif ication of N-hydroxy-AABP with acetate, or N-hydroxy-ABP with sulfate or acetate. How- ever, RNA adduct formation in cells exposed to either of these 2 compounds was unaffected by incubat ion in standard, sulfate-free, or acetate-fortif ied Hanks' solution (Chart 6).

DISCUSSION

The use of trit iated ABP derivatives of h igh specific activity allowed development of sensitive techniques for the detection of nucleic acid-bound carc inogen; RNA preparations containing in excess of 5 • 10 s d p m / m g RNA were obtained repeatedly. Incorporation of the acetyl group of N- hydroxy-AABP into RNA of isolated liver cells, as compared to binding of the aromatic nucleus to nucleic acid, was




3 .0 - N-hydroxy -AAF Protein

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N-hydroxy-AABP Protein

,O RNA I"NA I 0 . 5 ~

0 0.8 8 .0 0 0.8 8 .0

Sul fate Concen t ra t ion (mM) Chart 5. Influence of sulfate concentration on the incorporation of aryl-

hydroxamic acids into RNA and protein of isolated liver cells. Liver cells were incubated with 40 p.M N-hydroxy-['4C,3H]AAF or N-hydroxy-[~4C,SH]AABP with 0, 0.8, or 8.0 mM sulfate in the media. Open bars, ring label; closed bars, acetyl label.

lOO- t 0 ._ "a ~, 50 . Q.

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N-hydroxy-ABP

0 1 0 O 1 0

Acetate Concent ra t ion (mM)

0 . 8 0 .8 0 0

S u l f a t e Concent ra t ion tmMl

Chart 6. Influence of sulfate and acetate on the relative binding of N- hydroxy-AABP and N-hydroxy-ABP to RNA of isolated liver cells. Liver cells were incubated with 40 ~M N-hydroxy-[14C,3H]AABP or N-hydroxy-[3H]ABP with and without 0.8 mM sulfate or 10 mM acetate. The influence of sulfate and acetate on the relative ability of N-hydroxy-[14C,3H]AABP and N-hydroxy- [3H]ABP to form adducts with RNA was established by assigning the quantity of N-hydroxy-AABP incorporated into nucleic acid in 0.8 mM sulfate, the standard concentration in Hanks' solution, a value of 100, and expressing the relative aclduct formation in the other media as a percentage of this activity. Open bars, [3H]biphenyl; shaded bars, [14C]acetyl.

similar to data obtained from in viva exper iments (30). The low incorporat ion of the [carbonyl-~4C]acetyl group into protein or nucleic acid in the present exper iments demonstrated that incorporat ion of this label by convent ional bio- synthetic mechanisms was not a major considerat ion in the interpretation of these experiments.

Combination of the arylamine derivatives with RNA was chosen as an index of metabol ic activation of these cam-

Activat ion of Arylamines by Liver Cells

pounds since, in studies in viva with N-hydroxy-AABP and N-hydroxy-AAF, str iking dif ferences in the ratio of acetylated to deacetylated arylamine derivatives bound to RNA, but not DNA, have been described (17, 18, 28, 29). Analysis of the RNA adducts in the present study by UV spectroscopy, sucrose density gradient centr i fugat ion, and treatment with RNase or Pronase suggested that the label was not bound to contaminants of the nucleic acid. The low levels of radioact ivi ty bound to RNA that had been recovered from cells exposed to the labeled compounds for minimal periods demonstrated that the puri f icat ion techniques used in these studies were adequate, and that, unlike the binding of ni troso-BP to protein, these compounds did not react spontaneously with nucleic acid.

Combinat ion of N-hydroxy-AAF with RNA in these experiments undoubtedly resulted pr imari ly from conjugat ion of the hydroxamic acid with sulfate (Chart 7). This conclus ion is supported by both the sulfate-dependent b inding of the f luorene derivative to RNA and the acetylated form of the bound derivative. Al though the structures of the bound moieties have not been elucidated, it is improbable that label from tr i t iated acetate formed on hydrolysis of N-hydroxy-[14C,3H]AAF would have been incorporated into RNA. Most metabol ic pathways leading to incorporat ion of acetate into nucleic acid would lead to exchange of the t r i t ium; experiments in viva have borne this out (C. M. King and M. A. Shayman, unpubl ished observations). Moreover, [14C]acetate formed in the liver cells by hydrolysis of N- hydroxy-[14C,3H]AABP was incorporated to a much lesser extent than was the trit iated acetyl group of N-hydroxy- [14C,SH]AAF.

Of the biphenyl metabol i tes studied, N-hydroxy-AABP has been identif ied as the one most able to form adducts with RNA in liver cells. The available evidence suggests that N --, O acyltransfer (3, 21 ) was the pr imary mechanism responsible for the activation of this hydroxamic acid (Chart 7). The majori ty of the bound biphenyl derivatives did not retain the

?, N,-,,

Amine Amine

- - ~ ~ - N H a OH

o-Aminophenol

Nitro

O o-Quinone imine i! OH OH �9 _~.~.~j N= O

Hydroxamic acid 1 I ?l ~ 1 Hydroxylamine \,x~ ~ ~ ~ ' ~ " IN itroso O ~

\ ,zo., \ OX OX \ Arylamine- }roteir

Hydroxamic acid Hydroxylamine ~ \ adducts esters esters ~

Ary|amine-nucleic acid Arylacetamide-nucleic acid & protein adducts

& protein adducts

Chart 7. Possible mechanisms by which aminobiphenyl derivatives may be activated metabolically. Although biphenyl derivatives are shown here, some reactions have been demonstrated with other aromatic compounds. Names of the functional groups are given beneath the structures. The formation and disposition of N-oxidized amines has been reviewed recently (44). Possible esters of the hydroxamic acid or hydroxylamine are: sulfate, phos- phate, glucuronate, acetate, etc.

JULY 1976 2379



C. M. K ing et al.

acetyl group, and the substrate for acyltransferase, N-hydroxy-AABP, was bound to RNA more eff iciently than was any other derivative. AABP, an amide that requires only N- hydroxylat ion for conversion to an acyltransferase substrate, was the second most active compound in inducing adduct format ion. Unlike N-hydroxy-AAF, which did not appear to saturate the sulfate conjugat ion system at the highest concentrat ion util ized in our experiments (80 /zM), binding of N-hydroxy-AABP to RNA was not appreciably increased at concentrat ions of the hydroxamic acid greater than 30 /zM.

The conjugat ion of biphenyl derivatives with sulfate does not appear to play an important role in the activation of these compounds. Compared to N-hydroxy-AAF, sulfotransferase of rat liver cytosol is essentially ineffective in activating N-hydroxy-AABP. The concentrat ion of acetate or sulfate of the media did not seem to inf luence RNA adduct format ion from either N-hydroxy-ABP or N-hydroxy-AABP. Since N-hydroxy-AABP infrequently produces hepatomas (38), and N-hydroxy-AAF is a potent hepatocarcinogen (36), these observations are in agreement with previous studies that have correlated hepatocarcinogenic i ty with the activa- t ion of arylhydroxamic acids by conjugat ion with sulfate (8, 9, 11, 45-47). The mechanism(s) responsible for RNA adduct format ion in experiments with ABP, N-hydroxy-ABP, and nitroso-BP are unclear. Either the isolated cells must possess suff icient endogenous acetylation capacity to gen- erate suitable acyltransferase substrates (31, 33) or there must be other as yet unknown activation systems. Further experiments wil l be required to clarify this point.

It is not surprising that little adduct format ion occurred in incubat ions that contained nitro-BP, since the oxidizing condi t ions of the incubat ions were unfavorable for reduc- t ion of the nitro group, a reaction that may be required for activation of this compound (Chart 7). The low level of incorporat ion of azoxy-BP in these experiments decreased the probabi l i ty that this derivative, a breakdown product of N-hydroxy-ABP and nitroso-BP that is usually present as a minor impuri ty in preparations of these compounds, was responsible for the adduct format ion observed in experiments with these latter 2 derivatives.

The protein adducts formed in these experiments ac- counted for most of the labeled derivatives bound to tissue macromolecules. The specif ic activity of the protein was invariably higher than that of the RNA, and far more protein than RNA is present in the cells. Since no detergent was uti l ized in the homogenizat ion procedure, it is probable that the DNA remained with the protein in the phenol layer. The contr ibut ion of DNA to the radioactivity of the protein samples would be relatively insignif icant, however, because only small quantit ies would be present, and the level of b inding to DNA in vivo is low (18, 19, 28, 29).

The activation requirements for the format ion of protein adducts are less str ingent than those for nucleic acid adduct format ion. Metabol ical ly activated derivatives of arylamines that can react with nucleic acid, esters of hydroxamic acids (8, 9, 19, 25, 26), and hydroxylamines, (3, 5, 15) can also react wi th protein (25, 26) (Chart 7). Other metabolites, o-quinone imines (12, 22, 39) and nitroso derivatives (32), react with protein but not with nucleic acid (23) (Chart

7). Accordingly, the relatively greater incorporat ion of the ABP moiety into protein, compared with RNA, in short-term incubations of N-hydroxy-AABP with liver cells (Charts 2 and 3) probably reflects in part the reaction of nitroso-BP (32) formed fo l lowing deacetylation of the hydroxamic acid (14) and oxidat ion of the hydroxylamine (40). In experiments with the nitroso derivative, the extent of protein adduct formation is greatly dependent on the concentrat ion of the substrate. RNA adduct formation, on the other hand, is essentially unchanged above 25 /~M nitroso-BP and is pre- sumably l imited by the finite enzymatic capacity of the cell to activate the nitroso-BP. Consequently, it can be assumed that the metabol ic pathways that account for protein adduct format ion are more numerous than those that lead to nucleic acid adducts.

This report demonstrates that there are many advantages to using intact liver cells for the study of the metabolic activation of carcinogens. The cells were eff icient in inducing adduct format ion; greater than 14% of the compound incubated was bound to cellular macromolecules in some experiments. The compl icat ion of extrahepatic metabolism of the compounds was avoided, and discrete differences attr ibutable to the structures of the substrates were, there- fore, more readily obtained.

In some cases, as with the activation of arylhydroxamic acids by conjugat ion with sulfate, it was possible to define the role of cofactors without the int roduct ion of other organic compounds that could interfere with alternative metabolic pathways.

Through the use of isolated liver cells it should be possible to obtain highly labeled adducts for structural studies and to provide a means by which we can approach the metabolism of aromatic amines and other carcinogens as they relate to substrate specificity, enzyme induct ion, sex, strain, species, and transformation studies. In addit ion to the use of liver cells, these observations demonstrate the feasibil i ty of applying similar techniques to other susceptible tissues that, because of dif f icult ies in obtaining suffi- cient material, have received less attention than the liver.

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JULY 1976 2381



1976;36:2374-2381. Cancer Res Charles M. King, Nancy Raab Traub, Raymond A. Cardona, et al. Liver Parenchymal Cells2-Aminofluorene Derivatives with Macromolecules of Isolated Comparative Adduct Formation of 4-Aminobiphenyl and

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comparative adduct formation of 4-aminobiphenyl and 2 ... · an initial n-oxidation (7), followed...

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