[CANCER RESEARCH 49, 1038-1044, February15. 1989] Study of the Ability of Phenacetin, Acetaminophen, and Aspirin to Induce Cytotoxicity, Mutation, and Morphological Transformation in C3H/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 Research Institute, Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033

ABSTRACT commonly used nonprescription analgesic. To our knowledge there is no definitive experimental evidence linking aspirin Use of the analgesic compounds acetylsalicyclic acid (aspirin), phen- usage to human cancer induction. Aspirin is noncarcinogenic acetin, and acetaminophen has been correlated with increased risk of to rodents when administered alone even at doses that induce renal cancer in humans. Hence, we studied these compounds for ability chronic gastric irritation and ulcération(3). There is limited to induce cytotoxicity, mutation to ouabain resistance, and morphological transformation in cultured C3H/10T'/2 clone 8 (lOT'/i) mouse embryo evidence that chronic high doses of aspirin may be cocarcino- genic with MNNG4 in rat forestomach (3). This effect is prob cells. All three compounds were cytotoxic from 0.5-mg/ml to 2-mg/ml concentrations as evidenced by decreased plating efficiency. None of the ably related to the aspirin-induced erosion and hyperplasia compounds induced detectable base substitution mutations to ouabain sensitizing the gastric epithelium to MNNG. On the other resistance even at cytotoxic concentrations. Aspirin did not induce mor hand, aspirin may inhibit FANFT-induced urinary bladder le phological transformation. Both phenacetin and acetaminophen induced sions, possibly by inhibiting metabolism of FANFT by prosta- low but concentration-dependent numbers of atypical, weak type II mor glandin endoperoxide synthetase (4). Aspirin also inhibits TPA phologically transformed foci; at equimolar concentrations, phenacetin promotion of mouse skin carcinogenesis and blocks the TPA- was 1.1- to 3.0-fold more active in inducing these foci. Neither phenacetin nor acetaminophen was cotransforming with 3-methylcholanthrene, and dependent induction of ornithine decarboxylase activity in neither compound promoted cell transformation when added to 3-meth- mouse skin (reviewed in Ref. 5). ylcholanthrene-initiated 1111'; cells. The focus-inducing potency of both Phenacetin was widely used in humans as an analgesic and compounds was increased by addition of an Arochlor-induced hamster antipyretic drug, either alone or in combination with aspirin liver S9 fraction as an exogenous metabolizing system. However, seven and caffeine (reviewed in Refs. 2 and 6), but its use has been putative metabolites of phenacetin and acetaminophen that were tested— curtailed due to reports of toxic side effects. Long-term use of /V-hydrox\ phenacetin, /7-phenetidine, p-aminophenol, p-nitrosophenol, phenacetin, usually attributable to substance abuse, is associated benzoquinone, acetamide, and yV-acetyl-p-benzoquinoneimine—were in with kidney damage, renal carcinoma, and tumors of the bladder active in transformation assays at the concentrations reducing plating (2-8). Phenacetin was carcinogenic in long-term feeding studies efficiency of treated cells to 50% of the plating efficiency of nontreated (control) cells. Several acetaminophen- and phenacetin-induced foci were in C57BL/6 x C3H F, (hereafter called B6C3F,) mice (7) and Sprague-Dawley rats (8-10), inducing tumors of the kidney and cloned, expanded into cell lines, and characterized. These cell lines stably lower urinary tract. There is also limited evidence that 7V- formed type II foci when maintained at confluence for 2 to 4 wk in reconstruction experiments with nontransformed 10T'/2 cells; however, hydroxyphenacetin, a minor metabolite, is carcinogenic in rats they did not exhibit significantly increased saturation density compared (11). Bacterial mutagenesis assays have yielded conflicting re to 101 '/j cells, and they did not grow in soft agarose. These results sults (2, 12), but it is generally accepted that rodent liver suggest that metabolic intermediates of high concentrations of phenacetin microsomal and S9 fractions can activate phenacetin and 7V- and acetaminophen induce a low frequency of nonneoplastic morpholog hydroxyphenacetin to metabolites that are mutagenic to Sal ical transformation of lOT'A mouse embryo cells. monella tester strain TA 100 (13, 14). 7V-Hydroxyphenetidine and p-nitrosophenetol are thought to be the direct-acting mu tagenic metabolites (15-18). Hamster liver S9 activated phen INTRODUCTION acetin weakly in a forward mutation test in V79 Chinese ham There is now widespread interest in tentatively identifying ster cells (13). Phenacetin itself weakly induced chromosomal suspected human carcinogens through epidemiological investi aberrations in the absence and strongly induced them in the gations and confirming these observations by testing chemicals presence of rat liver S9 in Chinese hamster ovary cells (13). in studies of mutation and transformation of mammalian cells Acetaminophen, a widely used over-the-counter analgesic/ in vitro and in whole animal carcinogenesis bioassays (1). Re antipyretic, is safe at recommended dosages but can be toxic cently, epidemiological evidence has accumulated to suggest and fatal if ingested in large quantities. Overdoses of acetamin that use of certain analgesic compounds such as aspirin, phen ophen induce severe hepatic necrosis and renal failure (reviewed acetin and acetaminophen may be associated with increased in Ref. 19). Long-term rodent carcinogenesis bioassays have risk of renal cancer in humans (reviewed in Ref. 2). yielded conflicting results, since acetaminophen was carcino Aspirin (acetylsalicylic acid), a nonsteroidal inhibitor of pros- genic to liver in one study (20) but noncarcinogenic in two taglandin biosynthesis, is perhaps the most well-known and other studies (21, 22). Animals in all three studies had evidence of hepatic toxicity. Acetaminophen was nonmutagenic to bac 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 teria in the absence or presence of metabolic activating systems of page charges. This article must therefore be hereby marked advertisement in (16) and nongenotoxic to rat primary hepatocyte cultures (23). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by Grant ES-03816 from the National Institute of Environmental Much evidence implicates metabolites of phenacetin and Health Sciences, NIH, to J. R. L.; by Grant CA41277 from the National Cancer acetaminophen as the ultimate toxic and carcinogenic species Institute. NIH, to J. R. L., and by Grant SIG-2 from the American Cancer Society to B. E. H., and J. R. L. 4The abbreviations used are: MNNG, N-methyl-Af'-nitro-A'-nitrosoguanidine; 2Supported by an Individual Postdoctoral National Research Service Award. MCA, 3-methylcholanthrene; TPA, 12-O-tetradecanoylphorbol-13-acetate; Oua', Present address: Department of Pharmacology, George Washington University ouabain resistant; NABQI, A'-acetyl-p-benzoquinoneimine; FANFT, yV-[4-(F-ni- Medical School, Washington, DC 20037. tro-2-furyl)-2-thiazolyl]formamide; DMSO, dimethyl sulfoxide; LCOT,50% lethal 3To whom requests for reprints should be addressed. concentration. 1038 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN

(reviewed in Refs. 6, 19, and 24), and a schematic summary of stained with Giemsa, and viable colonies containing greater than 20 these reviews is provided here as Fig. 1. In general, in all animal cells were counted under a microscope. Mutation frequencies were species studied, approximately 70% of ingested phenacetin is calculated as follows. converted to acetaminophen via dealkylation by liver P450 total Ouar colonies/total no. of dishes Mutation frequency = enzymes. Furthermore, both phenacetin and acetaminophen plating efficiency of reseeded cells x 10* can undergo numerous ring hydroxylation reactions which are not shown in Fig. 1 and are not addressed in this study. Several Assays for Morphological Transformation. These assays were con of the metabolic intermediates of phenacetin and acetamino ducted exactly as described in 10T'/2 cells (30), except that the time of phen can form covalent adducts with cellular macromolecules treatment with compound was either 3 h in the presence of S9 fractions (6,19, 25), and others can generate free radicals and Superoxide or 24 h without S9 (38). In some experiments cells were treated (26, 27). For instance, para-phenetidine can form a homopoly- beginning on Day 5 after seeding according to the procedure of Nesnow mer free radical, jY-(4-ethoxyphenyl)benzoquinoneimine, and et al. to enhance sensitivity of the transformation assay (39). In these NABQI is a toxic intermediate common to both acetaminophen experiments, both type II and type III transformed colonies were scored (via epoxidation or acetaminophen free radical formation) and and tabulated (30; reviewed in Ref. 32). The cell inoculum treated in phenacetin (via A'-hydroxylation and hydrolysis of conjugation each assay was 2000 cells per 60-mm dish, and 20 dishes were treated products) (24). Formation of such reactive metabolites is for each concentration of compound studied per experiment. To bio thought to be critical to the toxicity, mutagenicity, and potential logically characterize the foci induced by various compounds, foci were ring cloned and expanded into cell lines. For analysis of growth rate carcinogenicity of these compounds. Therefore, the purpose of and saturation density, IO4cells were seeded per 60-mm cultures dish, this investigation was to (a) test the hypothesis that these four dishes for each time point, and allowed to grow with medium analgesics are carcinogenic, by studying several of these com changes every 3 days. Cells were harvested by trypsinization and pounds in assays for mutation and morphological transforma counted electronically on a Model Z Coulter Counter every 2 days for tion in cultured mammalian cells, and (b) to attempt to identify up to 20 days. Cells were tested for ability to grow in soft agarose using the potential carcinogenic intermediates by testing several me the original methods of MacPherson (40) modified by use of agarose tabolites of these compounds for in vitro cell transformation. instead of agar as per Boreiko and Heidelberger (41). Isolation of Hamster Liver S9. Arochlor 1254-induced hamster liver S9 was isolated from female Syrian golden hamsters (110 g) essentially MATERIALS AND METHODS as previously described for rat liver (38). Protein concentration was determined by Biorad assay to be 7 to 10 mg/ml of homogenate in 0.15 Chemicals. MCA, acetylsalicylic acid (aspirin), 4-nitrosophenol, p- M KC1, using bovine serum albumin as the standard. Homogenate was phenetidine, acetamide. benzoquinone, and 4-aminophenol were pur added to the activation mix and diluted 1:3 with serum-free medium to chased from Aldrich Chemical Company, Milwaukee, WI. Ouabain yield a final S9 protein concentration of 2 to 2.5 mg/ml as previously octahydrate, MNNG, TPA, acetaminophen, and phenacetin were pur described (38). This was empirically determined to be the maximum chased from Sigma Chemical Company, St. Louis, MO. Spectropho- noncytotoxic amount of S9 that cells could tolerate for 3 h. This tometric grade DMSO was purchased from the J. T. Baker Company, complete activation mixture was filtered through a 0.45-^m filter and Phillipsburg, NJ. Spectrophotometric grade acetone was purchased applied to cells in the absence or presence of test compound for 3 h from Mallinkrodt, Inc., Paris, KY. yV-Hydroxyphenacetin and NABQI (38). were generous gifts from Dr. Sidney Nelson, Department of Medicinal Chemistry, University of Washington (28). Cell Culture and Cytotoxicity Assays. The C3H/10T'/2 clone 8 RESULTS (10T'/2) mouse embryo fibroblast cell line, derived by Reznikoff et al. (29) and used in studies of chemical transformation (30, 31), was Standard Assays for Cytotoxicity, Mutagenesis, and Transfor cultured as previously described (29) with recent modifications detailing mation serum screening and medium preparation procedures (reviewed in Ref. 32), in medium without antibiotics. Cells were routinely screened for Acetylsalicylic Acid. The results of Cytotoxicity, mutagenesis, Mycoplasma by growth on Mycoplasma agar (33) and found to be and transformation experiments when lOT'/z cells were treated negative. Cytotoxicity assays were conducted as previously described with acetylsalicylic acid are summarized in Table 1. This com (30) and directly paralleled mutagenesis and transformation assays. pound decreased the plating efficiency in a dose-dependent Colonies containing greater than 20 cells were scored under a micro manner to 72, 32, and 25% of control values when 1.0, 2.0, and scope as viable. Assays for Mutation to Ouabain Resistance in OII/IOT1 Cells. 3.0 mg/ml, respectively, were applied to 10T'/2 cells for 24 h. These assays, which detect stable base substitution mutations to ouabain At these concentrations acetylsalicylic acid did not induce de resistance that map to mouse chromosome 3 (31, 34-36; reviewed in tectable mutation to ouabain resistance in experiments in which Ref. 37), were performed as previously described (31). Briefly, from ten MNNG induced an increase of 152-fold in mutation frequency, to twenty 100-mm dishes were each seeded with IO5 10T'/2 cells, and to 305 x 10~6. Table 1 also shows the cumulative data from 2 cells were treated 24 h later with 50 ^1 of a solution of MNNG in transformation assays utilizing the standard treatment regimen acetone, or of test compounds in DMSO, for 3 or 24 h. Following (31). Acetysalicylic acid induced no significant transformation treatment, the medium was removed and replaced with medium not in two experiments in which the positive control, 3-methyl- containing the compounds, and cells were incubated for a further 48 h, which yields the maximum expression time for MNNG and /V-acetox- cholanthrene, induced a total of 16 type II and type III foci. Based on these negative results, together with our unpublished yacetylaminofluorene-induced mutations to ouabain resistance in 1OT'/2 observations5 that aspirin actually inhibits 3-methylcholan- cells (31). Cells were then trypsinized, and twenty 100-mm dishes were each reseeded at an inoculum of 10s cells per 100-mm dish to assay threne-induced transformation, we did not investigate aspirin Ouar mutant colonies, and five 60-mm dishes were each seeded with further. 200 cells and five dishes with 2000 cells each, to assay plating efficiency Acetaminophen. The Cytotoxicity and genotoxicity of acet of replated cells as described above. Twenty-four h after reseeding, the aminophen are also summarized in Table 1. Treatment of 10T'/2 medium on dishes containing 10' cells per 100-mm dish was removed cells with 0.5, 1.0, and 2.0 mg/ml of acetaminophen caused a and replaced with medium containing 3 mM ouabain. Medium was dose-dependent Cytotoxicity, decreasing survival from 49% to removed and replaced with fresh medium containing 3 mM ouabain 9%, but did not induce detectable mutation to ouabain resist- again after 6 and 12 days of incubation. After 16 days of total incubation in ouabain, the Ouar mutant colonies were fixed with methanol and 5J. R. Landolph, manuscript in preparation. 1039 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. GENOTOXICITYOF PHENACETIN, ACETAMINOPHEN,AND ASPIRIN

Table 1 Summary of the activities of aspirin, phenacetin, and acetaminophen in standard genotoxicity assays dishes)6Type transformation (foci/total mutation with type II frequency 11 IIIfoci0/10052/1180/40 or III foci/total TreatmentAcetone, survival10084±21C96(x10')°23052 foci0/10035/1181/40 foci0/10087/118(14.7)''1/40 dishes0/10077/1181/40 0.5%MNNG

hMCA11 Mg/ml, 5

hAcetylsalicylictig/ml 24 ±1173"

acid 0.5 mg/ml, 24 h (0.5) 1.0 mg/ml, 24 h 72" 0/39 0/39 0/39 (0) 0/39 2.0 mg/ml, 24 h 32° 218 1/35 0/35 1/35 (0.57) 1/35 hAcetaminophen3.0 mg/ml, 24 25°49 0/202/40 0/200/40 0/20(0)2/40(1) 0/202/40

0.5 mg/ml, 24 h ±29 1.0 mg/ml, 24 h 16 ±24 2732 3/40 0/40 3/40(1.5) 3/40 hPhenacetin2.0 mg/ml, 24 9±11785 6/353/54 0/350/54 6/35(3.4)3/54(1.1) 6/353/54

0.5 mg/ml, 24 h ±2457 1.0 mg/ml, 24 h ±25 17/65 0/65 17/65 (4.6) 15/65 2.0 mg/ml', 24 h%of 45 ±19Ouar 3Cumulative 12/40Type 0/40Total 12/40(5.5)Dishes 11/40 " 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 low etin have promoter-like activity, cells were initiated with a low but concentration-dependent number of type II morphologically dose (0.1 jig/ml) of MCA for 24 h on Day 1 after seeding and transformed foci. For ease of comparison, the number of foci then exposed to acetaminophen or phenacetin for the duration normalized per 20 dishes is shown in parentheses in the column of the experiment beginning on Day 5 after seeding. Table 2 labeled total foci. shows that continuous exposure of cells initiated with 0.1 /ig/ Phenacetin. Treatment of 10T'/2 cells with 0.5, 1.0, and 2.0 ml of MCA to 0.125 Mg/ml of TPA increased focus formation mg/ml of phenacetin caused dose-dependent 15, 43, and 55% 3-fold. However, TPA did not significantly increase the fre decreases in plating efficiency, respectively (Table 1). Phenac quency or type of foci induced in cells initiated with 1 mg/ml etin was not detectably mutagenic at these concentrations but of acetaminophen or phenacetin. Table 2 also shows that con- did induce a dose-dependent increase in type II morphologically transformed foci. Phenacetin was 1.1-, 3.0-, and 1.6-fold more Table2 Assayofacetaminophenandphenacetinforabilitytoinitiateand promotecelltransformation potent than acetaminophen at concentrations of 0.5, 1.0, and III)°Withoutfoci (type II + 2.0 mg/ml, respectively. In addition, the transforming activity TreatmentInitiatorAcetone, of phenacetin occurred at much less cytotoxic concentrations pro than for acetaminophen (i.e., 5.5 foci/20 dishes at 45% survival promotion0/2013/202/403/40(1.5)'3/5417/65(4.6)2/20f2/202/202/202/202/202/20Withmotion1/202/20(2)3/20(3)6/20'0/201/202/190/204/203/19 0.5%MCA, 125Mg/mlTPATPATPATPATPAAcetaminophen, for phenacetin compared to 3.4 foci/20 dishes at 9% survival hAcetaminophen0.51.0 Mg/ml, 24 for acetaminophen at a concentration of 2 mg/ml). Phenacetin precipitated out of solution at a concentration of 2.0 mg/ml, likely accounting for the decrease in dose-response between the h1.0mg/ml, 24 hPhenacetin0.5mg/ml, 24 1.0- and 2.0-mg/ml treatment groups. To increase the sensitiv ity of the transformation assay we next tested the activity of h1.0mg/ml, 24 acetaminophen and phenacetin in the modified transformation hMCA0.mg/ml, 24 assay 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. h0.11 Mg/ml, 24 The number of MCA-induced foci/dish approximately doubled h0.1Mg/ml, 24 0.001mg/mlAcetaminophen, (1.9-fold increase) in the Nesnow assay compared to the stand ard assay; however, the transforming activity of acetaminophen h0.1Mg/ml, 24 0.005mg/mlAcetaminophen, and phenacetin was essentially unchanged in the Nesnow assay h0.1Mg/ml, 24 0.01mg/mlPhenacetin, compared to the standard transformation assay (data not h0.1Mg/ml, 24 0.001mg/mlPhenacetin, shown). h0.1Mg/ml, 24 0.005mg/mlPhenacetin, Initiation, Promotion, and Cocarcinogenesis Transformation As says Mg/ml, 24 hPromoterTPA,mg/mlTotal0.01 To determine whether the yield or type of foci induced by " 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 acetaminophen and phenacetin could be enhanced by tumor (1.0 Mg/ml)-TPA, 3 type II; Phenacetin (1.0 Mg/ml), 1 type II and 1 type III; promoters, 10T'/2 cells were treated on Day 1 with acetamino 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 phen or phenacetin and then exposed to 0.125 Mg/ml of TP A to a total of 20 dishes. for the duration of the experiment as per Mondai et al. (42). ' Significant to P < 0.05, MCA with TPA compared to MCA without TPA. Conversely, to determine whether acetaminophen and phenac Significant to P < 0.05, MCA with TPA compared to acetone with TPA. 1040 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN tinuous exposure of MCA-initiated cells to 0.001, 0.005, or Table 4 Transformation with S9 activation—standard assay/Nesnow 0.01 mg/ml of acetaminophen or phenacetin had no consistent Total foci (type II + type III)°/total effect on focus formation. Concentrations of acetaminophen dishes Standard Nesnow and phenacetin greater than 0.01 mg/ml actually inhibited %of sur- MCA-induced focus formation (not shown). Hence, acetamin Treatment vivai* Alone With S9 Alone With S9 ophen and phenacetin did not function as initiators or pro Acetone, 0.5% 100 0/40 0/40 0/40 1/40 moters of cell transformation in these assays. MCA We also determined whether acetaminophen and phenacetin 1 Mg/ml, 24 h 96 ±11' 11/40 ND* 46/40 ND were cotransforming with MCA by adding them simultaneously with MCA to cells for 24 h. Neither acetaminophen nor phen Cyclophosphamide acetin was synergistically cocarcinogenic with MCA (Table 3). 20 Mg/ml. 3 h 93 0/40 5/40 0/40 2/40 In fact, the highest concentration of acetaminophen (1.0 mg/ Acetaminophen ml) actually inhibited MCA transformation by 64%. Addition 0.5 mg/ml, 3 h 123(S9) 0/13 0/20 0/20 2/20 1.0 mg/ml, 3 h 108(89) 0/20 2/17 0/20 0/20 of a presumed ultimate cytotoxic metabolite, NABQI, did not increase but actually decreased MCA-induced transformation Phenacetin by 82% (Table 3). 0.5 mg/ml, 3 h 115 (S9) 0/20 0/20 0/15 4/15 1.0 mg/ml, 3h 88 (S9) 0/20 3/20 0/20 6/15 Metabolic Activation and Activity of Metabolites °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; To determine whether metabolic activation of acetaminophen cyclophosphamide, type II only; acetaminophen, type II only: phenacetin, type II and phenacetin was necessary for induction of foci by these only. Average of two experiments. Maximum average deviation was ±11%. compounds, we utilized a modification of the transformation ' Mean ±SEM of 3 or 4 experiments. The plating efficiency of untreated cells assay which incorporates a 3-h treatment in the presence of an ranged from 19 to 31%. S9 activation mix (38). Cyclophosphamide was included in ND, not determined. these experiments as a positive control for S9 activation, since our laboratory previously showed that metabolic activation is our own data with S9 activation indicated that metabolites of an absolute requirement for the transforming activity of this acetaminophen and phenacetin may be active toxic and carcin compound (38). We also compared the yield of S9-activated ogenic compounds, we next tested a battery of known, readily transformation between the standard assay and the Nesnow available metabolites in purified form for ability to induce assay. Table 4 shows data representative of several experiments. cytotoxicity and transformation. Fig. 1 shows the compounds The yield of MCA-induced transformed foci/plate increased tested (circled), and Fig. 2 shows the range of cytotoxicity for 4.2-fold in the Nesnow assay versus the standard assay. How each compound. All compounds induced dose-dependent cyto ever, the yield of transformed foci induced by S9-activated toxicity. The LC;o values varied greatly between compounds; cyclophosphamide did not increase but actually decreased by acetamide was the least cytotoxic metabolite, yielding a LC50 60% in the Nesnow assay compared to the standard assay. of approximately 300 mM. LCso values for the other compounds The yield of transformed foci following a 3-h treatment of were approximately 5 mM for acetaminophen, phenacetin, and p-phenetidine; 0.5 mM for /V-hydroxy-phenacetin; and 5 ¡Mifor cells with 1.0 mg/ml (but not 0.5 mg/ml) of acetaminophen or 4-aminophenol, benzoquinone, p-nitrosophenol, and TV-acetyl- phenacetin in the standard assay increased in the presence of p-benzoquinoneimine. The compounds were then tested at their S9 activation from 0 to 2 and 0 to 3 foci, respectively. In the Nesnow assay, S9 increased the yield of foci from 0 to 4 and 0 LCso for ability to induce morphological transformation using to 6 foci in cells treated with 0.5 mg/ml and 1.0 mg/ml of the modified transformation assay of Nesnow et al. (39) to phenacetin, respectively. The increases were less consistent with maximize sensitivity of detection. None of the metabolites S9-activated acetaminophen in the Nesnow assay, going from tested induced focus formation in these experiments (data not 0 and 0 foci at 0.5 and 1.0 mg/ml of acetaminophen without shown). S9 to 2 and 0 foci, respectively, with S9. Hence, S9 activation Biological Characterization of Cloned Transformed Foci was more positive for phenacetin in the Nesnow assay than for acetaminophen. Only type II foci were induced by acetamino To characterize the biological properties of acetaminophen- phen and phenacetin, even in the presence of S9 activation. and phenacetin-transformed cell lines, several foci induced by Since numerous literature reports (reviewed in Ref. 6) and each compound were cloned, expanded into cell lines, and studied. We noted that acetaminophen- and phenacetin-induced Table 3 Assay for possible cocarcinogenic effects between acetaminophen or phenacetin and MCA foci were usually significantly smaller than those induced by III)*Withoutfoci (type II + MCA but sufficiently large and distinct to clone from transfor mation assays (see Fig. 3 for representative foci). Four acet co- cocar- aminophen- and phenacetin-induced once cloned cell lines (la InitiatorAcetone, carcinogen0/4043/40cinogen13/38(6.8) beled ACT-1, ACT-2, PHN-1, and PHN-2) formed weak type 0.5% MCA0. II foci when passaged as nonsubcloned cultures and allowed to (2Tf remain confluent for 5 to 6 wk (Fig. 4). These four clones, 0.5 mg/ml 11/40(5.5) however, did not exhibit faster growth rates or increased satu Mg/ml Acetaminophen, 1.0 mg/ml 11/40(5.5) 4/40 (2.0) 0. Mg/ml Phenacetin, 0.5 mg/ml 11/40(5.5) 14/40 (7.0) ration densities compared to clone 8 cells and did not grow in 0. Mg/ml Phenacetin. 1.0 mg/ml 11/40(5.5) 16/39(8.2) soft agar (data not shown). Furthermore, continuous exposure 0. Mg/ml NABQI, 0.4 Mg/ml 11/40(5.5) 1/20(1.0) 0. Mg/mlTreatment"CocarcinogenAcetaminophen.NABQI, 1.3 Mg/mlTotal 11/40 (5.5)With 1/20(1.0) of these cell lines to 0.125 Mg/ml of TP A had only a moderate °Alltreatments 24 h. effect on the morphology of two of the four cell lines, no effects * The breakdown of the types of foci induced by the various treatments with on any of the growth parameters of any of the cell lines, and MCA was: MCA (1.0 Mg/ml), 26 type II and 17 type III; MCA (0.1 Mg/ml), 6 did not cause any of the clones to grow in soft agarose (data type II and 4 type III. ' Numbers in parentheses, total number of type II and type III foci normalized not shown). Attempts to enhance the differential properties to a total of 20 dishes. between phenacetin- and acetaminophen-induced transformed 1041 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN

gluewMdinon

Fig. 1. Metabolic scheme of phenacetin and acetaminophen compiled from the litera ture. The circled compounds were tested for transformation at their 50% lethal dose con centration.

WTOXIFICÕTK» TOXKfTY MUTAOCNtCITY CARCMOQEMOTY

». lili •¿â€¢:

IO'5 10'2 IO'1 10° 10 CONC (M)

Fig. 2. Cytotoxicity of various metabolites of acetaminophen and phenacetin P to C3H10T'/2 cells. D, p-aminophenol; O, benzoquinone; A, /»-nitrosophenol;V, yV-acetyl-p-benzoquinoneimine; T, /V-hydroxyphenacetin; •¿,p-phenetidine; •¿. acetaminophen; +. phenacetin; A, acetamide. 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) to CONTROL 3-MCA TPA.

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

W DISCUSSION The prevalent use, and potential for abuse, of all therapeutic ACETAMINOPHEN PHENACETIN drugs necessitates rigorous testing and study of such drugs for carcinogenic potential. The utility of the assay for morpholog ical transformation in C3H10T'/2 mouse embryo cells to detect and study mechanisms of action of chemical mutagens and carcinogens has been well documented (30, 31; reviewed in Refs. 32 and 37). Therefore, we used this assay to study phen acetin, acetaminophen, and aspirin for ability to cause cell transformation. Phenacetin has been implicated as a human Fig. 3. Macroscopic comparison of typical type II and type III foci induced by carcinogen of the lower urinary tract (2), and acetaminophen MCA to the weak type II foci induced by acetaminophen and phenacetin. might be suspected to have similar carcinogenic potential since it is a major metabolite of phenacetin in many animal species, clones and lOT'/z cells by subcloning the former were not has a related metabolic scheme (reviewed in Ref. 6), and has successful, because their colony morphology at low cell density activity in some animal carcinogenicity studies (20-22). Aspirin was not distinguishable from lOT'/z cells. This is in contrast to is not known to be carcinogenic to humans or experimental MCA-induced foci, which were easily cloned and then sub- animals and was inactive in our studies, except that it was 1042 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN cytotoxic at high concentrations. Therefore, this discussion will purified quinoneimine NABQI was highly cytotoxic but did not focus on acetaminophen and phenacetin. induce transformation by itself and did not enhance the trans Both phenacetin and acetaminophen are nephrotoxic and formation induced by low concentrations (0.1 ng/ml) of MCA. hepatotoxic in vivo at high doses, and these toxicities have been It is possible that, while the postulated product of the P450- observed in patients with tumors thought to be caused by drug mediated metabolism of acetaminophen and phenacetin (i.e., abuse (reviewed in Ref. 2). Thus, we first determined the cyto NABQI) was cytotoxic but not carcinogenic, by-products of the toxic concentration ranges of these drugs to use for mutation reaction (i.e., active oxygen species) may be ultimate carcino and transformation assays. The therapeutic p.o. doses of these genic species (27, 28, 46). It is also possible that extracellular drugs range from 500 to 1000 mg daily, and humans ingesting NABQI may not reach oncogenically relevant sites and may 1000 mg of acetaminophen have maximum blood levels of 30 require generation in the vicinity of DNA in order to transform to 40 Mg/ml of serum (43). Abusers of these drugs probably cells. Interestingly, Dybing et al. (47) showed that NABQI have much higher blood levels, and since 80 to 90% of these induced DNA single strand breaks in Reuber hepatoma cells drugs is conjugated and excreted in the urine, the local (neph- but was unable to cause mutations in Salmonella typhimurium. ron) concentration may be much higher than the blood level. Historically in this laboratory approximately 50% of typical We found that 0.5 to 2.0 mg/ml (500 to 2000 Mg/ml, 5 to 10 type II foci are tumorigenic in nude mice, whereas 80% of type mM) were required to induce cytotoxicity in lOT'/i cells. We III foci are tumorigenic (30; reviewed in Refs. 32 and 37). Since note that the concentrations of 3 to 7 mM acetaminophen that foci induced by acetaminophen and phenacetin were atypical were required to cause cytotoxicity in primary hepatocyte cul (weak) type II foci, it was important for us to clone and tures (23) are close to our values of 5 to 10 mM in 10T'/2 cells. characterize these foci. None of the foci cloned once from Another useful aspect of the mammalian lOT'/i transforma transformation assays could be recloned on the basis of trans tion assay is the ability to measure mutation to ouabain resist formed colony morphology. Even though the mixed clones ance in the same cells used to measure morphological transfor reformed weak type II foci when maintained at confluence, they mation. Indeed, for some chemicals such as benzo(a)pyrene, N- did not exhibit any other classical parameters of neoplastic acetoxy-acetylaminofluorene, and MCA, there is a good corre transformation, such as increased saturation density or anchor lation between concentrations that cause mutation and those age independence. While the significance of these types of foci that cause transformation (31). In our studies neither aspirin, is currently unknown, they may represent an early stage of cell acetaminophen, nor phenacetin caused base substitution mu transformation. However, the relative unresponsiveness of the tations to ouabain resistance even at highly cytotoxic concen cell lines to TPA indicates that the morphologically trans trations. formed phenotype of these cells would represent a very early, While bacterial mutagenesis systems are predominantly used non-TPA-responsive stage of transformation. for mutagenesis testing, approximately 55% of the tested car In summary we have shown that: (a) aspirin was completely cinogens are nonmutagenic (1). In this light, it is important nongenotoxic to C3H/10T'/2 cells; and (b) acetaminophen and that the lOT'A cell transformation system is effective at detect phenacetin were nonmutagenic, but S9-mediated metabolism ing some nonmutagenic organic carcinogens, such as 5-aza- of both of these compounds resulted in the induction of weak cytidine (35, 44), and nonmutagenic inorganic carcinogens, morphological transformation that was nonneoplastic in na such as nickel and arsenic compounds.6 Using the lOTVi cell ture. None of seven putative metabolic end-products of these transformation assay, we found that both acetaminophen and compounds induced transformation. These results are consist phenacetin, but not aspirin, induced a low, but dose-dependent, ent with the epidemiológica! evidence presented in the accom number of type II morphologically transformed foci. Interest panying paper by Ross et al. (48), showing that ingestion of ingly, increasing the number of cells at risk by using the modi various combinations of these analgesics and caffeine resulted fied protocol of Nesnow et al. (39) for treatment had no in small (approximately 2-fold) increases in the risk of renal significant effect on the yield of transformed foci except for S9- pelvic cancer. We suggest that, if acetaminophen and phenace activated phenacetin. The yield of transformed foci induced by tin are carcinogenic to humans, either other factors such as another nonmutagenic carcinogen, nickel sulfide, was similarly endogenous promoters or cocarcinogens not studied here act to unaffected by increasing the number of cells at risk.6 Further further transform the weakly transformed cells induced by more, acetaminophen and phenacetin were inactive as initiators phenacetin or acetaminophen, or other metabolic schemes such of TPA-promoted cell transformation, as promoters of MCA- as peroxidase or prostaglandin synthase-mediated catalysis (47, initiated cells, and as cotransforming agents with MCA. Since 49), or other mechanisms such as the recently demonstrated many investigators tend to equate initiation with mutation, it recruitment of activated macrophages to the site of toxicity (50, may not be surprising that these nonmutagenic compounds 51), might play an important role in tumor induction. were poor initiators and cocarcinogens, since they were unable to induce base-substitution mutations (Table 1). As described in the "Introduction," the pharmacological and ACKNOWLEDGMENTS toxicological activity of these compounds is related to a com The authors would like to thank Dr. Sidney Nelson, Department of plex metabolic scheme outlined in Fig. 1. Since C3H/10T'/2 Medicinal Chemistry, University of Washington, for generous gifts of A'-hydroxyphenacetin and A'-acetyl-p-benzoquinoneimine; Dr. Paul cells have retained P448 activity (reviewed in Refs. 32 and 37) but lack yV-hydroxylase activity (45), and since both parent Hochstein of the Institute for Toxicity and Dr. Ronald Ross, Dr. Dave Garabrant, and Dr. Mimi C. Yu of the Department of Preventive compounds were weakly active without exogenous metabolic activation, our results in C3H/10T'/2 cells are consistent with Medicine at the University of Southern California for helpful criticism of the manuscript; and Sarah Olivo and Gloria Barreras for typing the the hypothesis that the ultimate toxic intermediate is a qui- manuscript. noneimine generated by hydrolysis of conjugation products or via P450-mediated epoxidation (reviewed in Ref. 6). However, REFERENCES ' T. Miura, S. R. Patierno. and J. R. Landolph, Environmental and Molecular Mutagenesis, in press, 1989, and C. Troesch, and J. R. Landolph, manuscripts 1. Tennant, R. W., Margolin, B. H., Shelby, M. D., Zeiger, E., Haseman, J. K., in preparation. Spalding, J., Caspary, W., Resnick, M., Stasiewicz, S., Anderson, B., and 1043 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. GENOTOXICITY OF PHENACETIN, ACETAMINOPHEN, AND ASPIRIN

Minor, R. Prediction of chemical carcinogenicity in rodents from in vitro 26. Rosen, G. M., Singletary, U. V., Jr., Rauckman, E. J., and Killenberg, P. G. genetic toxicity assays. Science (Wash. DC), 236:933-941, 1987. Acetaminophen hepatotoxicity: an alternative mechanism. Biochem. Phar 2. Evaluation of carcinogenic risk of chemicals to man. Some pharmaceutical macol., 32:1701-1706, 1983. drugs. IARC Monogr., 24: 135-161, 1980. 27. Thelen, M., and Wendel, A. Drug-induced lipid peroxidation in mice. V. 3. Tsung-Hsien, C, Yu-Chung, L., Kwang-Yung, L., Cheng-Hsien, S., and Yee- Ethane production and glutathione release in the isolated liver upon perfusion Ping, C. Cocarcinogenic action of aspirin on gastric tumors induced by N- with acetaminophen. Biochem. Pharmacol., 32:1701-1706, 1983. nitroso-;V-meIhylnitroguanidine in rats. J. Nati. Cancer Inst., 70:1067-1075, 28. Dahlin, C., and Nelson, S. D. Synthesis, decomposition kinetics, and prelim 1973. inary toxicological studies of pure W-acetyl-p-benzoquinoneimine, a proposed 4. Cohen, S. M., Zenser, T. U., Marasaki, G., Fukushima, S., Mattammal, M. toxic metabolite of acetaminophen. J. Med. Chem., 25:805-895, 1982. B., Rapp, N. S., and Davis, B. B. Aspirin inhibition of JV-[4-(5-nitro-2-furyl)- 29. Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. Establishment and 2-thiazoly 1jformamide-induced lesions of the urinary bladder correlated with characterization of a cloned line of C3H mouse embryo cells sensitive to inhibition of metabolism by bladder prostaglandin endoperoxide synthetase. postconfluence inhibition of division. Cancer Res., 33: 3231-3238, 1973. Cancer Res., 41: 3355-3359, 1981. 30. Reznikoff, C. A., Bertram, J. S., Brankow, D. W., and Heidelberger, C. 5. Honn, K. V., Bockman, R. S., and Manien. L. J. Prostaglandins and cancer: Quantitative and qualitative studies of chemical transformation of cloned a review of tumor initiation through tumor metastasis. Prostaglandins, 21: C3H mouse cells sensitive to postconfluence inhibition of cell division. 833-864, 1981. Cancer Res., 33: 3239-3249, 1973. 6. Hinson, J. A. Reactive metabolites of phenacetin and acetaminophen: a 31. Landolph, J. R., and Heidelberger, C. Chemical carcinogens produce muta review. Environ. Health Perspect., 49: 71-79, 1983. tions to ouabain resistance in transformable C3H/10T'/2 Cl 8 mouse embryo 7. Nakanishi, K., Kurata, Y., Oshima, M., Fukushima, S., and Ito, N. Carcin fibroblasts. Proc. Nati. Acad. Sci. USA, 76: 930-934, 1979. ogenicity of phenacetin: long-term feeding study on B6OI-, mice. Int. J. 32. Landolph, J. R. Chemical transformation in C3H/10T'/2 Cl 8 mouse embryo Cancer, 29:434-444, 1982. cells: historical background, assessment of the transformation assay, and 8. Isaka, H., Yoshii, H., Otsuji, A., Koike, M., Nagai, T., Koura,. M., Sugiyasu, evolution and optimization of the transformation assay protocol. In: T. K., and Kanabayashi, T. Tumors of Sprague-Dawley rats induced by long- Kakanaga and H. Yamasaki (eds.), Transformation Assay of Established Cell term feeding of phenacetin. Gann, 70: 29-36, 1979. Lines: Mechanism and Application, pp. 185-198. Lyon: International 9. Johansson, S. Carcinogenicity of analgesics: long-term treatment of Sprague- Agency for Cancer Research, 1985. Dawley rats with phenacetin, phenazone, caffeine, and paracetamol (acet 33. Barile, M. F. Mycoplasma infections of cell cultures. Isr. J. Med. Sci., 17: aminophen). Int. J. Cancer, 27: 521-529, 1981. 555-562, 1981. 10. Johansson, S., Angervall, L., Bengtsson, U., and Wahlquist, L. Uroepithelial 34. Landolph, J. R., Telfer, N., and Heidelberger, C. Further evidence that tumors of the renal pelvis associated with abuse of phenacetin-containing ouabain resistant variants induced by chemical carcinogens in transformable analgesics. Cancer (Phila.), 33: 743-753, 1974. C3H/10T'/2 Cl 8 mouse fibroblasts are mutants. Mutât.Res., 72: 295-310, 11. Calder, I. C., Goss, D. E., Williams, P. J., Funder, C. C., Green, C. R., Harn, 1980. K. N., and Tange, J. D. Neoplasia in the rat induced by ¿V-hydroxyphenacetin, 35. Landolph, J. R., and Jones, P. A. Mutagenicity of 5-azacytidine and related a metabolite of phenacetin. Pathology, 8: 1-6, 1976. nucleosides in C3H/10T'/2 clone 8 and V79 cells. Cancer Res., «.-817-823, 12. Dunkel, U. C., Zeiger, E., Brusick, D., McCoy, E., McGregor, D., Mortal- 1982. mans, K., Rosenkrantz, H. S., and Simmon, V. F. Reproducibility of micro- 36. Landolph, J. R., and Fournier, R. E. K. Micron.'!! mediated transfer of bial mutagenicity assays. II. Testing of carcinogens and noncarcinogens in carcinogen-induced ouabain resistance from C3H/10T'/2 Cl 8 mouse fibro Salmonella typhímuríumandEscherichia coli. Environ. Mut., 7(suppl.): 1- blasts to human cells. Mutât.Res., 707: 447-463, 1983. 248, 1985. 37. Landolph, J. R. Mechanisms of chemically induced multistep neoplastic 13. DeFlora, S., Russo, P., Pala, M., Fassina, G., Zunino, A., Bennicelli, C., transformation in C3H/10T'/2 cells. In: E. Huberman and S. H. Barr (eds.), Zanacchi, P., Comoirano, A., and Parodi, S. Assay of phenacetin genotoxicity Carcinogenesis, Vol. 10, pp. 211-223. New York: Raven Press, 1985. using in vitro and in vivo test systems. J. Toxicol. Environ. Health, 16: 355- 38. Billings, P. C., Heidelberger, C., and Landolph, J. R. S-9 metabolic activation 377, 1985. enhances aflatoxin-mediated transformation of C3H/10T'/2 cells. Toxicol. 14. Camus, A., Friesen, M., Croisy, A., and Bartsch, H. Species-specific activa Appi. Pharmacol., 77: 58-65, 1985. tion of phenacetin into bacterial mutagen by hamster liver enzymes and 39. Nesnow, S., Garland, H., and Curtis, G. Improved transformation of C3H/ identification of jV-hydroxyphenacetin O-glucuronide as a promutagen in the 10T'/2 cells by direct- and indirect-acting carcinogens. Carcinogenesis urine. Cancer Res., 42: 3201-3209, 1982. (Lond.), 3:377-380,1980. 15. Glowinski, I. B., Sanderson, N. D., Hayashi, S., and Thorgeirsson, S. S. 40. MacPherson, I. Soft agar techniques. In: P. F. Krue, Jr., and M. K. Petterson, Metabolic activation and genotoxicity of jV-hydroxy-2-acetylaminofluorene Jr. (eds.), Tissue Culture: Methods and Applications, pp. 226-283. New and yv-hydroxyphenacetin derivatives in Reuber (H4-II-E) hepatoma cells. York; Academic Press, 1973. Cancer Res., 44: 1098-1104, 1984. 41. Boreiko, C., and Heidelberger, C. Isolation of mutants temperature-sensitive 16. Wirth, P. J., Dybing, E., von Bahr, C., and Thorgeirsson, S. S. Mechanism for expression of the transformed state from chemically transformed C3H/ of yv-hydroxyacetylarylamine mutagenicity in the Salmonella test system: 10T'/2 cells. Carcinogenesis (Lond.), /: 1059-1073, 1980. metabolic activation of JV-hydroxyphenacetin by liver and kidney fractions 42. Mondai, S., Brankow, D. M., and Heidelberger, C. Two-stage chemical from the rat, mouse, hamster, and man. Mol. Pharmacol., 18: 117-127, Carcinogenesis in cultures of C3H/10T'/2 cells. Cancer Res., 36: 2254-2260, 1980. 1976. 17. Wirth, P. H., Alewood, P., Calder, I., and Thorgeirsson, S. S. Mutagenicity 43. Kohli, U., Sherma, S. K., Azarwal, S. S., and Sahib, M. K. Influence of of JV-hydroxyphenacetin and their respective deacetylated metabolites in nutritional status on acetaminophen metabolism in man. Indian J. Med. Res., 75: 265-273, 1982. nitroreductase deficient Salmonella TA98FR and TA100FR. Carcinogenesis (Lond.), 3:167-170, 1982. 44. Benedict, W. F., Banerjee, A., Gardner, A., and Jones, P. Induction of morphological transformation in mouse C3H/10T'/2 clone 8 cells and chro 18. Mulder, G. J., Kadlubar, F. F., Mays, J. B., and Hinson, J. A. Reaction of mosome damage in hamster A(Ti) CI-3 cells by cancer chemotherapeutic mutagenic phenacetin metabolites with glutathione and DNA: possible im plications for toxicity. Mol. Pharmacol., 26: 342-347, 1984. agents. Cancer Res., 37:2202-2208, 1977. 45. Oglesby, L. A., Cudak, C., Curtis, G., Garland, H., Hyatt, B., Montgomery, 19. Hinson, J. A. Biochemical toxicology of acetaminophen. Biochem. Toxicol., L., and Nesnow, S. Relationship of A'-arylacetamide metabolism and mac- 2: 103-129, 1980. romolecular binding to oncogenic transformation of C3H10T'/2 Cl 8 cells. 20. Flaks, A., and Flaks, F. Induction of liver cell tumors in IF mice by para Cancer Lett., 16: 231-237, 1982. cetamol. Carcinogenesis (Lond.), 4: 363-368, 1983. 46. Mason, R. P., and Fisher, V. Free radicals of acetaminophen: their subsequent 21. Hiraga, K., and Fujii, T. Carcinogenicity testing of acetaminophen in F344 reactions and toxicological significance. Fed. Proc., 45: 2453-2499, 1986. rats. J. Cancer Res., 76: 79-89, 1985. 47. Dybing, E., Holme, J. A., Gordon, W. P., Soderlund, E. J., Dahlin, D. C., 22. Hagiwara, A., and Ward, J. M. The chronic hepatotoxic, tumor-promoting, and Nelson, S. D. Genotoxicity studies with paracetamol. Mutât.Res., 138: and carcinogenic effects of acetaminophen in male B6C3Fi mice. Fund. 21-32, 1984. Appi. Toxicol., 7: 376-386, 1986. 48. Ross, R. K., Paganini I HI!, A., Landolph, J., Gerkins, V., and Henderson, 23. Milam, K. M., and Byard, J. L. Acetaminophen metabolism, cytotoxicity, B. E. Analgesics, cigarette smoking, and other risk factors for cancer of the and genotoxicity in rat primary hepatocyte cultures. Toxicol. Appi. Phar renal pelvis and ureter. Cancer Res., 49:1045-1048, 1989. macol., 79: 342-347, 1985. 49. Potter, D. W., Miller, A. W., and Hinson, J. A. Identification of acetamin 24. Dahlin, D. C., Miwa, G. T., Lu, A. Y. H., and Nelson, S. D. A'-Acetyl-p- ophen polymerization products catalyzed by horseradish peroxidase. J. Biol. benzoquinoneimine: a cytochrome I' 450 mediated oxidation product of Chem., 260: 12174-12180, 1985. acetaminophen. Proc. Nati. Acad. Sci. USA, 81: 1327-1331, 1984. 50. Laskin, D. L., and Pilaro, A. M. Potential role of activated macrophages in 25. Gillette, J. R., Nelson, S. D., Mulder, G. J., Jollow, D. J., Mitchell, J. R., acetaminophen hepatotoxicity. I. Isolation and characterization of activated Pohl, L. R., and Hinson, J. A. Formation of chemically reactive metabolites macrophages from rat liver. Toxicol. Appi. Pharmacol., 86:204-215, 1986. of phenacetin and acetaminophen. In: R. Snyder, D. V. Parke, J. J. Kocsis, 51. Laskin, D. L., Pilar, A. M., and Ji, S. Potential role of activated macrophages C. G. Gibson, and C. M. Witmer (eds.), Biological Reactive Intermediates, in acetaminophen hepatoxicity. II. Mechanism of macrophage accumulation Vol. 2, pp. 931-950. New York: Plenum, 1982. and activation. Toxicol. Appi. Pharmacol., 86: 216-266, 1986.

1044 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. Study of the Ability of Phenacetin, Acetaminophen, and Aspirin to Induce Cytotoxicity, Mutation, and Morphological Transformation in C3H/10T½ Clone 8 Mouse Embryo Cells

Steven R. Patierno, Norman L. Lehman, Brian E. Henderson, et al.

Cancer Res 1989;49:1038-1044.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/49/4/1038

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/49/4/1038. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research.