[CANCER RESEARCH 49, 1038-1044, February15. 1989]

Study of the Ability of Phenacetin, Acetaminophen, and Aspirin to InduceCytotoxicity, Mutation, and Morphological Transformation inC3H/10TV2 Clone 8 Mouse Embryo Cells1

Steven R. Patierno,2 Norman L. Lehman, Brian E. Henderson, and Joseph R. Landolph3

Departments of Microbiology and Pathology fS. R. P., N. L. L., J. R. L.] and Preventive Medicine [B. E. H.J, Kenneth Norris, Jr., Cancer Hospital and ResearchInstitute, Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033

ABSTRACT

Use of the analgesic compounds acetylsalicyclic acid (aspirin), phen-

acetin, and acetaminophen has been correlated with increased risk ofrenal cancer in humans. Hence, we studied these compounds for abilityto induce cytotoxicity, mutation to ouabain resistance, and morphologicaltransformation in cultured C3H/10T'/2 clone 8 (lOT'/i) mouse embryo

cells. All three compounds were cytotoxic from 0.5-mg/ml to 2-mg/ml

concentrations as evidenced by decreased plating efficiency. None of thecompounds induced detectable base substitution mutations to ouabainresistance even at cytotoxic concentrations. Aspirin did not induce morphological transformation. Both phenacetin and acetaminophen inducedlow but concentration-dependent numbers of atypical, weak type II mor

phologically transformed foci; at equimolar concentrations, phenacetinwas 1.1- to 3.0-fold more active in inducing these foci. Neither phenacetinnor acetaminophen was cotransforming with 3-methylcholanthrene, andneither compound promoted cell transformation when added to 3-meth-ylcholanthrene-initiated 1111'; cells. The focus-inducing potency of both

compounds was increased by addition of an Arochlor-induced hamster

liver S9 fraction as an exogenous metabolizing system. However, sevenputative metabolites of phenacetin and acetaminophen that were tested—/V-hydrox\ phenacetin, /7-phenetidine, p-aminophenol, p-nitrosophenol,benzoquinone, acetamide, and yV-acetyl-p-benzoquinoneimine—were in

active in transformation assays at the concentrations reducing platingefficiency of treated cells to 50% of the plating efficiency of nontreated(control) cells. Several acetaminophen- and phenacetin-induced foci were

cloned, expanded into cell lines, and characterized. These cell lines stablyformed type II foci when maintained at confluence for 2 to 4 wk inreconstruction experiments with nontransformed 10T'/2 cells; however,

they did not exhibit significantly increased saturation density comparedto 101 '/j cells, and they did not grow in soft agarose. These results

suggest that metabolic intermediates of high concentrations of phenacetinand acetaminophen induce a low frequency of nonneoplastic morphological transformation of lOT'A mouse embryo cells.

INTRODUCTION

There is now widespread interest in tentatively identifyingsuspected human carcinogens through epidemiological investigations and confirming these observations by testing chemicalsin studies of mutation and transformation of mammalian cellsin vitro and in whole animal carcinogenesis bioassays (1). Recently, epidemiological evidence has accumulated to suggestthat use of certain analgesic compounds such as aspirin, phenacetin and acetaminophen may be associated with increasedrisk of renal cancer in humans (reviewed in Ref. 2).

Aspirin (acetylsalicylic acid), a nonsteroidal inhibitor of pros-taglandin biosynthesis, is perhaps the most well-known and

Received 3/11/88; revised 10/7/88; accepted 11/16/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by Grant ES-03816 from the National Institute of Environmental

Health Sciences, NIH, to J. R. L.; by Grant CA41277 from the National CancerInstitute. NIH, to J. R. L., and by Grant SIG-2 from the American CancerSociety to B. E. H., and J. R. L.

2Supported by an Individual Postdoctoral National Research Service Award.

Present address: Department of Pharmacology, George Washington UniversityMedical School, Washington, DC 20037.

3To whom requests for reprints should be addressed.

commonly used nonprescription analgesic. To our knowledgethere is no definitive experimental evidence linking aspirinusage to human cancer induction. Aspirin is noncarcinogenicto rodents when administered alone even at doses that inducechronic gastric irritation and ulcération(3). There is limitedevidence that chronic high doses of aspirin may be cocarcino-genic with MNNG4 in rat forestomach (3). This effect is probably related to the aspirin-induced erosion and hyperplasiasensitizing the gastric epithelium to MNNG. On the otherhand, aspirin may inhibit FANFT-induced urinary bladder lesions, possibly by inhibiting metabolism of FANFT by prosta-glandin endoperoxide synthetase (4). Aspirin also inhibits TPApromotion of mouse skin carcinogenesis and blocks the TPA-dependent induction of ornithine decarboxylase activity inmouse skin (reviewed in Ref. 5).

Phenacetin was widely used in humans as an analgesic andantipyretic drug, either alone or in combination with aspirinand caffeine (reviewed in Refs. 2 and 6), but its use has beencurtailed due to reports of toxic side effects. Long-term use ofphenacetin, usually attributable to substance abuse, is associatedwith kidney damage, renal carcinoma, and tumors of the bladder(2-8). Phenacetin was carcinogenic in long-term feeding studiesin C57BL/6 x C3H F, (hereafter called B6C3F,) mice (7) andSprague-Dawley rats (8-10), inducing tumors of the kidney andlower urinary tract. There is also limited evidence that 7V-hydroxyphenacetin, a minor metabolite, is carcinogenic in rats(11). Bacterial mutagenesis assays have yielded conflicting results (2, 12), but it is generally accepted that rodent livermicrosomal and S9 fractions can activate phenacetin and 7V-hydroxyphenacetin to metabolites that are mutagenic to Salmonella tester strain TA 100 (13, 14). 7V-Hydroxyphenetidineand p-nitrosophenetol are thought to be the direct-acting mutagenic metabolites (15-18). Hamster liver S9 activated phenacetin weakly in a forward mutation test in V79 Chinese hamster cells (13). Phenacetin itself weakly induced chromosomalaberrations in the absence and strongly induced them in thepresence of rat liver S9 in Chinese hamster ovary cells (13).

Acetaminophen, a widely used over-the-counter analgesic/antipyretic, is safe at recommended dosages but can be toxicand fatal if ingested in large quantities. Overdoses of acetaminophen induce severe hepatic necrosis and renal failure (reviewedin Ref. 19). Long-term rodent carcinogenesis bioassays haveyielded conflicting results, since acetaminophen was carcinogenic to liver in one study (20) but noncarcinogenic in twoother studies (21, 22). Animals in all three studies had evidenceof hepatic toxicity. Acetaminophen was nonmutagenic to bacteria in the absence or presence of metabolic activating systems(16) and nongenotoxic to rat primary hepatocyte cultures (23).

Much evidence implicates metabolites of phenacetin andacetaminophen as the ultimate toxic and carcinogenic species

4The abbreviations used are: MNNG, N-methyl-Af'-nitro-A'-nitrosoguanidine;MCA, 3-methylcholanthrene; TPA, 12-O-tetradecanoylphorbol-13-acetate; Oua',ouabain resistant; NABQI, A'-acetyl-p-benzoquinoneimine; FANFT, yV-[4-(F-ni-tro-2-furyl)-2-thiazolyl]formamide; DMSO, dimethyl sulfoxide; LCOT,50% lethalconcentration.

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GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN

(reviewed in Refs. 6, 19, and 24), and a schematic summary ofthese reviews is provided here as Fig. 1. In general, in all animalspecies studied, approximately 70% of ingested phenacetin isconverted to acetaminophen via dealkylation by liver P450enzymes. Furthermore, both phenacetin and acetaminophencan undergo numerous ring hydroxylation reactions which arenot shown in Fig. 1 and are not addressed in this study. Severalof the metabolic intermediates of phenacetin and acetaminophen can form covalent adducts with cellular macromolecules(6,19, 25), and others can generate free radicals and Superoxide(26, 27). For instance, para-phenetidine can form a homopoly-mer free radical, jY-(4-ethoxyphenyl)benzoquinoneimine, andNABQI is a toxic intermediate common to both acetaminophen(via epoxidation or acetaminophen free radical formation) andphenacetin (via A'-hydroxylation and hydrolysis of conjugation

products) (24). Formation of such reactive metabolites isthought to be critical to the toxicity, mutagenicity, and potentialcarcinogenicity of these compounds. Therefore, the purpose ofthis investigation was to (a) test the hypothesis that theseanalgesics are carcinogenic, by studying several of these compounds in assays for mutation and morphological transformation in cultured mammalian cells, and (b) to attempt to identifythe potential carcinogenic intermediates by testing several metabolites of these compounds for in vitro cell transformation.

MATERIALS AND METHODS

Chemicals. MCA, acetylsalicylic acid (aspirin), 4-nitrosophenol, p-phenetidine, acetamide. benzoquinone, and 4-aminophenol were purchased from Aldrich Chemical Company, Milwaukee, WI. Ouabainoctahydrate, MNNG, TPA, acetaminophen, and phenacetin were purchased from Sigma Chemical Company, St. Louis, MO. Spectropho-tometric grade DMSO was purchased from the J. T. Baker Company,Phillipsburg, NJ. Spectrophotometric grade acetone was purchasedfrom Mallinkrodt, Inc., Paris, KY. yV-Hydroxyphenacetin and NABQIwere generous gifts from Dr. Sidney Nelson, Department of MedicinalChemistry, University of Washington (28).

Cell Culture and Cytotoxicity Assays. The C3H/10T'/2 clone 8(10T'/2) mouse embryo fibroblast cell line, derived by Reznikoff et al.

(29) and used in studies of chemical transformation (30, 31), wascultured as previously described (29) with recent modifications detailingserum screening and medium preparation procedures (reviewed in Ref.32), in medium without antibiotics. Cells were routinely screened forMycoplasma by growth on Mycoplasma agar (33) and found to benegative. Cytotoxicity assays were conducted as previously described(30) and directly paralleled mutagenesis and transformation assays.Colonies containing greater than 20 cells were scored under a microscope as viable.

Assays for Mutation to Ouabain Resistance in OII/IOT1 Cells.

These assays, which detect stable base substitution mutations to ouabainresistance that map to mouse chromosome 3 (31, 34-36; reviewed inRef. 37), were performed as previously described (31). Briefly, from tento twenty 100-mm dishes were each seeded with IO5 10T'/2 cells, and

cells were treated 24 h later with 50 ^1 of a solution of MNNG inacetone, or of test compounds in DMSO, for 3 or 24 h. Followingtreatment, the medium was removed and replaced with medium notcontaining the compounds, and cells were incubated for a further 48 h,which yields the maximum expression time for MNNG and /V-acetox-yacetylaminofluorene-induced mutations to ouabain resistance in 1OT'/2cells (31). Cells were then trypsinized, and twenty 100-mm dishes wereeach reseeded at an inoculum of 10s cells per 100-mm dish to assayOuar mutant colonies, and five 60-mm dishes were each seeded with

200 cells and five dishes with 2000 cells each, to assay plating efficiencyof replated cells as described above. Twenty-four h after reseeding, themedium on dishes containing 10' cells per 100-mm dish was removed

and replaced with medium containing 3 mM ouabain. Medium wasremoved and replaced with fresh medium containing 3 mM ouabainagain after 6 and 12 days of incubation. After 16 days of total incubationin ouabain, the Ouar mutant colonies were fixed with methanol and

stained with Giemsa, and viable colonies containing greater than 20cells were counted under a microscope. Mutation frequencies werecalculated as follows.

Mutation frequency =total Ouar colonies/total no. of dishes

plating efficiency of reseeded cells x 10*

Assays for Morphological Transformation. These assays were conducted exactly as described in 10T'/2 cells (30), except that the time of

treatment with compound was either 3 h in the presence of S9 fractionsor 24 h without S9 (38). In some experiments cells were treatedbeginning on Day 5 after seeding according to the procedure of Nesnowet al. to enhance sensitivity of the transformation assay (39). In theseexperiments, both type II and type III transformed colonies were scoredand tabulated (30; reviewed in Ref. 32). The cell inoculum treated ineach assay was 2000 cells per 60-mm dish, and 20 dishes were treatedfor each concentration of compound studied per experiment. To biologically characterize the foci induced by various compounds, foci werering cloned and expanded into cell lines. For analysis of growth rateand saturation density, IO4cells were seeded per 60-mm cultures dish,

four dishes for each time point, and allowed to grow with mediumchanges every 3 days. Cells were harvested by trypsinization andcounted electronically on a Model Z Coulter Counter every 2 days forup to 20 days. Cells were tested for ability to grow in soft agarose usingthe original methods of MacPherson (40) modified by use of agaroseinstead of agar as per Boreiko and Heidelberger (41).

Isolation of Hamster Liver S9. Arochlor 1254-induced hamster liverS9 was isolated from female Syrian golden hamsters (110 g) essentiallyas previously described for rat liver (38). Protein concentration wasdetermined by Biorad assay to be 7 to 10 mg/ml of homogenate in 0.15M KC1, using bovine serum albumin as the standard. Homogenate wasadded to the activation mix and diluted 1:3 with serum-free medium toyield a final S9 protein concentration of 2 to 2.5 mg/ml as previouslydescribed (38). This was empirically determined to be the maximumnoncytotoxic amount of S9 that cells could tolerate for 3 h. Thiscomplete activation mixture was filtered through a 0.45-^m filter andapplied to cells in the absence or presence of test compound for 3 h(38).

RESULTS

Standard Assays for Cytotoxicity, Mutagenesis, and Transformation

Acetylsalicylic Acid. The results of Cytotoxicity, mutagenesis,and transformation experiments when lOT'/z cells were treated

with acetylsalicylic acid are summarized in Table 1. This compound decreased the plating efficiency in a dose-dependentmanner to 72, 32, and 25% of control values when 1.0, 2.0, and3.0 mg/ml, respectively, were applied to 10T'/2 cells for 24 h.

At these concentrations acetylsalicylic acid did not induce detectable mutation to ouabain resistance in experiments in whichMNNG induced an increase of 152-fold in mutation frequency,to 305 x 10~6. Table 1 also shows the cumulative data from 2

transformation assays utilizing the standard treatment regimen(31). Acetysalicylic acid induced no significant transformationin two experiments in which the positive control, 3-methyl-cholanthrene, induced a total of 16 type II and type III foci.Based on these negative results, together with our unpublishedobservations5 that aspirin actually inhibits 3-methylcholan-threne-induced transformation, we did not investigate aspirinfurther.

Acetaminophen. The Cytotoxicity and genotoxicity of acetaminophen are also summarized in Table 1. Treatment of 10T'/2

cells with 0.5, 1.0, and 2.0 mg/ml of acetaminophen caused adose-dependent Cytotoxicity, decreasing survival from 49% to9%, but did not induce detectable mutation to ouabain resist-

5J. R. Landolph, manuscript in preparation.

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Table 1 Summary of the activities of aspirin, phenacetin, and acetaminophen in standard genotoxicity assays

TreatmentAcetone,

0.5%MNNG

1 Mg/ml, 5hMCA1

tig/ml 24hAcetylsalicylic

acid0.5 mg/ml, 24 h1.0 mg/ml, 24 h2.0 mg/ml, 24 h3.0 mg/ml, 24hAcetaminophen

0.5 mg/ml, 24 h1.0 mg/ml, 24 h2.0 mg/ml, 24hPhenacetin

0.5 mg/ml, 24 h1.0 mg/ml, 24 h2.0 mg/ml', 24 h%of

survival10084±21C96

±1173"

72"32°

25°49

±2916 ±249±11785

±2457±25

45 ±19Ouar

mutation

frequency(x10')°23052

218

2732

3Cumulative

transformation (foci/totaldishes)6Type

11foci0/10035/1181/40

0/391/350/202/40

3/406/353/54

17/6512/40Type

IIIfoci0/10052/1180/40

0/390/350/200/40

0/400/350/54

0/650/40Total

foci0/10087/118(14.7)''1/40

(0.5)0/39 (0)1/35 (0.57)0/20(0)2/40(1)

3/40(1.5)6/35(3.4)3/54(1.1)

17/65 (4.6)12/40(5.5)Dishes

with type IIor III foci/total

dishes0/10077/1181/40

0/391/350/202/40

3/406/353/54

15/6511/40

" Average of 2 experiments.b Cumulative transformation of 2 to 5 experiments using 20 dishes per point.' Mean ±SEM of 3 to 4 experiments except where noted; plating efficiency of untreated cells ranged from 19 to 31%.d Numbers in parentheses, total number of foci normalized on the basis of 20 dishes.' Precipitates out of solution at this concentration.

ance. However, acetaminophen did consistently induce a lowbut concentration-dependent number of type II morphologicallytransformed foci. For ease of comparison, the number of focinormalized per 20 dishes is shown in parentheses in the columnlabeled total foci.

Phenacetin. Treatment of 10T'/2 cells with 0.5, 1.0, and 2.0mg/ml of phenacetin caused dose-dependent 15, 43, and 55%decreases in plating efficiency, respectively (Table 1). Phenacetin was not detectably mutagenic at these concentrations butdid induce a dose-dependent increase in type II morphologicallytransformed foci. Phenacetin was 1.1-, 3.0-, and 1.6-fold morepotent than acetaminophen at concentrations of 0.5, 1.0, and2.0 mg/ml, respectively. In addition, the transforming activityof phenacetin occurred at much less cytotoxic concentrationsthan for acetaminophen (i.e., 5.5 foci/20 dishes at 45% survivalfor phenacetin compared to 3.4 foci/20 dishes at 9% survivalfor acetaminophen at a concentration of 2 mg/ml). Phenacetinprecipitated out of solution at a concentration of 2.0 mg/ml,likely accounting for the decrease in dose-response between the1.0- and 2.0-mg/ml treatment groups. To increase the sensitivity of the transformation assay we next tested the activity ofacetaminophen and phenacetin in the modified transformationassay of Nesnow et al. (39), in which 10T'/2 cells were seeded

at 2000 cells/dish and treated for 24 h beginning on Day 5.The number of MCA-induced foci/dish approximately doubled(1.9-fold increase) in the Nesnow assay compared to the standard assay; however, the transforming activity of acetaminophenand phenacetin was essentially unchanged in the Nesnow assaycompared to the standard transformation assay (data notshown).

Initiation, Promotion, and Cocarcinogenesis Transformation Assays

To determine whether the yield or type of foci induced byacetaminophen and phenacetin could be enhanced by tumorpromoters, 10T'/2 cells were treated on Day 1 with acetamino

phen or phenacetin and then exposed to 0.125 Mg/ml of TP Afor the duration of the experiment as per Mondai et al. (42).Conversely, to determine whether acetaminophen and phenac

etin have promoter-like activity, cells were initiated with a lowdose (0.1 jig/ml) of MCA for 24 h on Day 1 after seeding andthen exposed to acetaminophen or phenacetin for the durationof the experiment beginning on Day 5 after seeding. Table 2shows that continuous exposure of cells initiated with 0.1 /ig/ml of MCA to 0.125 Mg/ml of TPA increased focus formation3-fold. However, TPA did not significantly increase the frequency or type of foci induced in cells initiated with 1 mg/mlof acetaminophen or phenacetin. Table 2 also shows that con-

Table2 Assayofacetaminophenandphenacetinfor abilityto initiateandpromotecelltransformation

TreatmentInitiatorAcetone,

0.5%MCA,1.0 Mg/ml, 24hAcetaminophen0.5

mg/ml, 24h1.0mg/ml, 24hPhenacetin0.5

mg/ml, 24h1.0mg/ml, 24hMCA0.

1 Mg/ml, 24h0.1Mg/ml, 24h0.1

Mg/ml, 24h0.1

Mg/ml, 24h0.1

Mg/ml, 24h0.1

Mg/ml, 24h0.1

Mg/ml, 24 hPromoterTPA,

125Mg/mlTPATPATPATPATPAAcetaminophen,

0.001mg/mlAcetaminophen,

0.005mg/mlAcetaminophen,

0.01mg/mlPhenacetin,

0.001mg/mlPhenacetin,

0.005mg/mlPhenacetin,

0.01mg/mlTotal

foci (type II +III)°Without

promotion0/2013/202/403/40(1.5)'3/5417/65(4.6)2/20f2/202/202/202/202/202/20Withpro

motion1/202/20(2)3/20(3)6/20'0/201/202/190/204/203/19

" The breakdown of the types of foci induced by the various treatments was:acetone-TPA, 1 type II; MCA (1.0 Mg/ml), 9 type II and 4 type III; Acetaminophen(1.0 Mg/ml)-TPA, 3 type II; Phenacetin (1.0 Mg/ml), 1 type II and 1 type III;MCA (0.1 Mg/ml)-TPA, 4 type 11and 2 type III.

b Numbers in parentheses, total number of type II and type III foci normalized

to a total of 20 dishes.' Significant to P < 0.05, MCA with TPA compared to MCA without TPA.

Significant to P < 0.05, MCA with TPA compared to acetone with TPA.

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GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN

tinuous exposure of MCA-initiated cells to 0.001, 0.005, or0.01 mg/ml of acetaminophen or phenacetin had no consistenteffect on focus formation. Concentrations of acetaminophenand phenacetin greater than 0.01 mg/ml actually inhibitedMCA-induced focus formation (not shown). Hence, acetaminophen and phenacetin did not function as initiators or promoters of cell transformation in these assays.

We also determined whether acetaminophen and phenacetinwere cotransforming with MCA by adding them simultaneouslywith MCA to cells for 24 h. Neither acetaminophen nor phenacetin was synergistically cocarcinogenic with MCA (Table 3).In fact, the highest concentration of acetaminophen (1.0 mg/ml) actually inhibited MCA transformation by 64%. Additionof a presumed ultimate cytotoxic metabolite, NABQI, did notincrease but actually decreased MCA-induced transformationby 82% (Table 3).

Metabolic Activation and Activity of Metabolites

To determine whether metabolic activation of acetaminophenand phenacetin was necessary for induction of foci by thesecompounds, we utilized a modification of the transformationassay which incorporates a 3-h treatment in the presence of anS9 activation mix (38). Cyclophosphamide was included inthese experiments as a positive control for S9 activation, sinceour laboratory previously showed that metabolic activation isan absolute requirement for the transforming activity of thiscompound (38). We also compared the yield of S9-activatedtransformation between the standard assay and the Nesnowassay. Table 4 shows data representative of several experiments.The yield of MCA-induced transformed foci/plate increased4.2-fold in the Nesnow assay versus the standard assay. However, the yield of transformed foci induced by S9-activatedcyclophosphamide did not increase but actually decreased by60% in the Nesnow assay compared to the standard assay.

The yield of transformed foci following a 3-h treatment ofcells with 1.0 mg/ml (but not 0.5 mg/ml) of acetaminophen orphenacetin in the standard assay increased in the presence ofS9 activation from 0 to 2 and 0 to 3 foci, respectively. In theNesnow assay, S9 increased the yield of foci from 0 to 4 and 0to 6 foci in cells treated with 0.5 mg/ml and 1.0 mg/ml ofphenacetin, respectively. The increases were less consistent withS9-activated acetaminophen in the Nesnow assay, going from0 and 0 foci at 0.5 and 1.0 mg/ml of acetaminophen withoutS9 to 2 and 0 foci, respectively, with S9. Hence, S9 activationwas more positive for phenacetin in the Nesnow assay than foracetaminophen. Only type II foci were induced by acetaminophen and phenacetin, even in the presence of S9 activation.

Since numerous literature reports (reviewed in Ref. 6) and

Table 3 Assay for possible cocarcinogenic effects between acetaminophen orphenacetin and MCA

Table 4 Transformation with S9 activation—standard assay/NesnowTotal foci (type II + type III)°/total

dishes

InitiatorAcetone,

0.5%MCA0.

Mg/ml0. Mg/ml0. Mg/ml0. Mg/ml0. Mg/mlTreatment"CocarcinogenAcetaminophen.

0.5 mg/mlAcetaminophen, 1.0 mg/mlPhenacetin, 0.5 mg/mlPhenacetin. 1.0 mg/mlNABQI, 0.4 Mg/mlNABQI, 1.3 Mg/mlTotal

foci (type II +III)*Without

co-carcinogen0/4043/40

(2Tf11/40(5.5)11/40(5.5)11/40(5.5)11/40(5.5)11/40(5.5)11/40 (5.5)With

cocar-cinogen13/38(6.8)

4/40 (2.0)14/40 (7.0)16/39(8.2)

1/20(1.0)1/20(1.0)

°All treatments 24 h.* The breakdown of the types of foci induced by the various treatments with

MCA was: MCA (1.0 Mg/ml), 26 type II and 17 type III; MCA (0.1 Mg/ml), 6type II and 4 type III.

' Numbers in parentheses, total number of type II and type III foci normalized

to a total of 20 dishes.

Treatment

Standard Nesnow%of sur-

vivai* Alone With S9 Alone With S9

Acetone, 0.5% 100 0/40 0/40 0/40 1/40

MCA1 Mg/ml, 24 h 96 ±11' 11/40 ND* 46/40 ND

93 0/40 5/40 0/40 2/40Cyclophosphamide

20 Mg/ml. 3 h

Acetaminophen0.5 mg/ml, 3 h 123(S9) 0/13 0/20 0/20 2/201.0 mg/ml, 3 h 108(89) 0/20 2/17 0/20 0/20

Phenacetin0.5 mg/ml, 3 h 115 (S9) 0/20 0/20 0/15 4/151.0 mg/ml, 3h 88 (S9) 0/20 3/20 0/20 6/15

°The breakdown of the types of foci induced in each treatment group was:

MCA standard, 7 type II and 4 type III; Nesnow, 22 type II and 24 type III;cyclophosphamide, type II only; acetaminophen, type II only: phenacetin, type IIonly.

Average of two experiments. Maximum average deviation was ±11%.' Mean ±SEM of 3 or 4 experiments. The plating efficiency of untreated cells

ranged from 19 to 31%.ND, not determined.

our own data with S9 activation indicated that metabolites ofacetaminophen and phenacetin may be active toxic and carcinogenic compounds, we next tested a battery of known, readilyavailable metabolites in purified form for ability to inducecytotoxicity and transformation. Fig. 1 shows the compoundstested (circled), and Fig. 2 shows the range of cytotoxicity foreach compound. All compounds induced dose-dependent cyto

toxicity. The LC;o values varied greatly between compounds;acetamide was the least cytotoxic metabolite, yielding a LC50of approximately 300 mM. LCso values for the other compoundswere approximately 5 mM for acetaminophen, phenacetin, andp-phenetidine; 0.5 mM for /V-hydroxy-phenacetin; and 5 ¡Mifor4-aminophenol, benzoquinone, p-nitrosophenol, and TV-acetyl-p-benzoquinoneimine. The compounds were then tested at their

LCso for ability to induce morphological transformation usingthe modified transformation assay of Nesnow et al. (39) tomaximize sensitivity of detection. None of the metabolitestested induced focus formation in these experiments (data notshown).

Biological Characterization of Cloned Transformed Foci

To characterize the biological properties of acetaminophen-and phenacetin-transformed cell lines, several foci induced by

each compound were cloned, expanded into cell lines, andstudied. We noted that acetaminophen- and phenacetin-induced

foci were usually significantly smaller than those induced byMCA but sufficiently large and distinct to clone from transformation assays (see Fig. 3 for representative foci). Four acetaminophen- and phenacetin-induced once cloned cell lines (labeled ACT-1, ACT-2, PHN-1, and PHN-2) formed weak type

II foci when passaged as nonsubcloned cultures and allowed toremain confluent for 5 to 6 wk (Fig. 4). These four clones,however, did not exhibit faster growth rates or increased saturation densities compared to clone 8 cells and did not grow insoft agar (data not shown). Furthermore, continuous exposureof these cell lines to 0.125 Mg/ml of TP A had only a moderateeffect on the morphology of two of the four cell lines, no effectson any of the growth parameters of any of the cell lines, anddid not cause any of the clones to grow in soft agarose (datanot shown). Attempts to enhance the differential propertiesbetween phenacetin- and acetaminophen-induced transformed

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GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN

gluewMdinon

Fig. 1. Metabolic scheme of phenacetinand acetaminophen compiled from the literature. The circled compounds were tested fortransformation at their 50% lethal dose concentration.

WTOXIFICÕTK» TOXKfTY MUTAOCNtCITY

CARCMOQEMOTY

10 IO'5

CONC (M)

10'2 IO'1 10°

Fig. 2. Cytotoxicity of various metabolites of acetaminophen and phenacetinto C3H10T'/2 cells. D, p-aminophenol; O, benzoquinone; A, /»-nitrosophenol;V,yV-acetyl-p-benzoquinoneimine; T, /V-hydroxyphenacetin; •¿�,p-phenetidine; •¿�.acetaminophen; +. phenacetin; A, acetamide.

CONTROL 3-MCA

WACETAMINOPHEN PHENACETIN

Fig. 3. Macroscopic comparison of typical type II and type III foci induced byMCA to the weak type II foci induced by acetaminophen and phenacetin.

clones and lOT'/z cells by subcloning the former were not

successful, because their colony morphology at low cell densitywas not distinguishable from lOT'/z cells. This is in contrast toMCA-induced foci, which were easily cloned and then sub-

».

lili•¿�•:

P

Fig. 4. Response of cell lines cloned from foci induced by acetaminophen(ACT-1 and ACT-2), phenacetin (PHN-1 and PHN-2), or MCA (MNL-1) toTPA.

cloned because of their dramatically altered morphology compared to that of 10T'/2 cells.

DISCUSSION

The prevalent use, and potential for abuse, of all therapeuticdrugs necessitates rigorous testing and study of such drugs forcarcinogenic potential. The utility of the assay for morphological transformation in C3H10T'/2 mouse embryo cells to detect

and study mechanisms of action of chemical mutagens andcarcinogens has been well documented (30, 31; reviewed inRefs. 32 and 37). Therefore, we used this assay to study phenacetin, acetaminophen, and aspirin for ability to cause celltransformation. Phenacetin has been implicated as a humancarcinogen of the lower urinary tract (2), and acetaminophenmight be suspected to have similar carcinogenic potential sinceit is a major metabolite of phenacetin in many animal species,has a related metabolic scheme (reviewed in Ref. 6), and hasactivity in some animal carcinogenicity studies (20-22). Aspirinis not known to be carcinogenic to humans or experimentalanimals and was inactive in our studies, except that it was

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GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN

cytotoxic at high concentrations. Therefore, this discussion willfocus on acetaminophen and phenacetin.

Both phenacetin and acetaminophen are nephrotoxic andhepatotoxic in vivo at high doses, and these toxicities have beenobserved in patients with tumors thought to be caused by drugabuse (reviewed in Ref. 2). Thus, we first determined the cytotoxic concentration ranges of these drugs to use for mutationand transformation assays. The therapeutic p.o. doses of thesedrugs range from 500 to 1000 mg daily, and humans ingesting1000 mg of acetaminophen have maximum blood levels of 30to 40 Mg/ml of serum (43). Abusers of these drugs probablyhave much higher blood levels, and since 80 to 90% of thesedrugs is conjugated and excreted in the urine, the local (neph-ron) concentration may be much higher than the blood level.We found that 0.5 to 2.0 mg/ml (500 to 2000 Mg/ml, 5 to 10mM) were required to induce cytotoxicity in lOT'/i cells. We

note that the concentrations of 3 to 7 mM acetaminophen thatwere required to cause cytotoxicity in primary hepatocyte cultures (23) are close to our values of 5 to 10 mM in 10T'/2 cells.

Another useful aspect of the mammalian lOT'/i transforma

tion assay is the ability to measure mutation to ouabain resistance in the same cells used to measure morphological transformation. Indeed, for some chemicals such as benzo(a)pyrene, N-acetoxy-acetylaminofluorene, and MCA, there is a good correlation between concentrations that cause mutation and thosethat cause transformation (31). In our studies neither aspirin,acetaminophen, nor phenacetin caused base substitution mutations to ouabain resistance even at highly cytotoxic concentrations.

While bacterial mutagenesis systems are predominantly usedfor mutagenesis testing, approximately 55% of the tested carcinogens are nonmutagenic (1). In this light, it is importantthat the lOT'A cell transformation system is effective at detecting some nonmutagenic organic carcinogens, such as 5-aza-cytidine (35, 44), and nonmutagenic inorganic carcinogens,such as nickel and arsenic compounds.6 Using the lOTVi cell

transformation assay, we found that both acetaminophen andphenacetin, but not aspirin, induced a low, but dose-dependent,number of type II morphologically transformed foci. Interestingly, increasing the number of cells at risk by using the modified protocol of Nesnow et al. (39) for treatment had nosignificant effect on the yield of transformed foci except for S9-activated phenacetin. The yield of transformed foci induced byanother nonmutagenic carcinogen, nickel sulfide, was similarlyunaffected by increasing the number of cells at risk.6 Further

more, acetaminophen and phenacetin were inactive as initiatorsof TPA-promoted cell transformation, as promoters of MCA-initiated cells, and as cotransforming agents with MCA. Sincemany investigators tend to equate initiation with mutation, itmay not be surprising that these nonmutagenic compoundswere poor initiators and cocarcinogens, since they were unableto induce base-substitution mutations (Table 1).

As described in the "Introduction," the pharmacological and

toxicological activity of these compounds is related to a complex metabolic scheme outlined in Fig. 1. Since C3H/10T'/2

cells have retained P448 activity (reviewed in Refs. 32 and 37)but lack yV-hydroxylase activity (45), and since both parentcompounds were weakly active without exogenous metabolicactivation, our results in C3H/10T'/2 cells are consistent withthe hypothesis that the ultimate toxic intermediate is a qui-noneimine generated by hydrolysis of conjugation products orvia P450-mediated epoxidation (reviewed in Ref. 6). However,

' T. Miura, S. R. Patierno. and J. R. Landolph, Environmental and Molecular

Mutagenesis, in press, 1989, and C. Troesch, and J. R. Landolph, manuscriptsin preparation.

purified quinoneimine NABQI was highly cytotoxic but did notinduce transformation by itself and did not enhance the transformation induced by low concentrations (0.1 ng/ml) of MCA.It is possible that, while the postulated product of the P450-mediated metabolism of acetaminophen and phenacetin (i.e.,NABQI) was cytotoxic but not carcinogenic, by-products of thereaction (i.e., active oxygen species) may be ultimate carcinogenic species (27, 28, 46). It is also possible that extracellularNABQI may not reach oncogenically relevant sites and mayrequire generation in the vicinity of DNA in order to transformcells. Interestingly, Dybing et al. (47) showed that NABQIinduced DNA single strand breaks in Reuber hepatoma cellsbut was unable to cause mutations in Salmonella typhimurium.

Historically in this laboratory approximately 50% of typicaltype II foci are tumorigenic in nude mice, whereas 80% of typeIII foci are tumorigenic (30; reviewed in Refs. 32 and 37). Sincefoci induced by acetaminophen and phenacetin were atypical(weak) type II foci, it was important for us to clone andcharacterize these foci. None of the foci cloned once fromtransformation assays could be recloned on the basis of transformed colony morphology. Even though the mixed clonesreformed weak type II foci when maintained at confluence, theydid not exhibit any other classical parameters of neoplastictransformation, such as increased saturation density or anchorage independence. While the significance of these types of fociis currently unknown, they may represent an early stage of celltransformation. However, the relative unresponsiveness of thecell lines to TPA indicates that the morphologically transformed phenotype of these cells would represent a very early,non-TPA-responsive stage of transformation.

In summary we have shown that: (a) aspirin was completelynongenotoxic to C3H/10T'/2 cells; and (b) acetaminophen andphenacetin were nonmutagenic, but S9-mediated metabolismof both of these compounds resulted in the induction of weakmorphological transformation that was nonneoplastic in nature. None of seven putative metabolic end-products of thesecompounds induced transformation. These results are consistent with the epidemiológica! evidence presented in the accompanying paper by Ross et al. (48), showing that ingestion ofvarious combinations of these analgesics and caffeine resultedin small (approximately 2-fold) increases in the risk of renalpelvic cancer. We suggest that, if acetaminophen and phenacetin are carcinogenic to humans, either other factors such asendogenous promoters or cocarcinogens not studied here act tofurther transform the weakly transformed cells induced byphenacetin or acetaminophen, or other metabolic schemes suchas peroxidase or prostaglandin synthase-mediated catalysis (47,49), or other mechanisms such as the recently demonstratedrecruitment of activated macrophages to the site of toxicity (50,51), might play an important role in tumor induction.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Sidney Nelson, Department ofMedicinal Chemistry, University of Washington, for generous gifts ofA'-hydroxyphenacetin and A'-acetyl-p-benzoquinoneimine; Dr. Paul

Hochstein of the Institute for Toxicity and Dr. Ronald Ross, Dr. DaveGarabrant, and Dr. Mimi C. Yu of the Department of PreventiveMedicine at the University of Southern California for helpful criticismof the manuscript; and Sarah Olivo and Gloria Barreras for typing themanuscript.

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1989;49:1038-1044. Cancer Res   Steven R. Patierno, Norman L. Lehman, Brian E. Henderson, et al.   Transformation in C3H/10T½ Clone 8 Mouse Embryo Cellsto Induce Cytotoxicity, Mutation, and Morphological Study of the Ability of Phenacetin, Acetaminophen, and Aspirin

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