2CL8 cells

Mutation Research, 222 (1989) 223-235

223

Elsevier MTR 01375

DNA adduct formation, metabolism, and morphological transforming activity of aceanthrylene in C3H10T1/2CL8 cells * S. Nesnow 1, j. Ross 1, N. M o h a p a t r a 1,, ,, A. Gold 3, R. Sangaiah 3 and R. Gupta 2 t Carcinogenesis and Metabolism Branch, U.S. Em~ironmental Protection Agency, Research Triangle Park, NC 27711, 2 Department of Pharmacology, Baylor College of Medicine Houston, TX, 77030, and ~ Department of Enc'ironmental Sciences and Engineering, Uniuersity of North Carolina at Chapel Hill, Chapel Hill, NC 27514 (U. S. A.)

(Received 2 March 1988) (Revision received 20 September 1988) (Accepted 25 September 1988)

Keyword~: Aceanth~'lene; Morphologicaltransformation; C3H10T1/2 cells; DNA adducts; 32P-postlabeling

Summao' Aceanthrylenc (ACE), a cyclopenta-fused polycyclic aromatic hydrocarbon (CP-PAH) related to anthracene, has been studied for its ability to be metabolized, to form D N A adducts, and to morphologically transform C 3 H 1 0 T 1 / 2 C L 8 mouse embryo fibroblasts in culture. Although ACE has been previously shown to be a strong mutagen in S a l m o n e l l a t y p h i m u r i u m strains TA89 and TA100, it did not transform C 3 H I O T 1 / 2 cells (0.4-16 # g / m l ) under 2 treatment protocols: treatment (for 24 h) 1 day after seeding the cells; treatment (for 24 h) 5 days after seeding the cells. Both protocols are effective in detecting the morphological transforming activity of PAH and C P - P A H and the latter protocol has been shown to be effective in detecting chemicals which are active in the first protocol only with the additional treatment of the cells with a tumor promoter. ACE is mctabolized by C 3 H 1 0 T 1 / 2 cells to ACE-l,2-dihydrodiol (the cyclopenta-ring dihydrodiol) at a rate of 450 pmoles ACE-1,2-dihydrodiol f o r m e d / h / 1 0 6 cells. ACE-7,8dihydrodiol and ACE-9,10-dihydrodiol, identified as major Aroclor-1254-induced rat liver microsomal metabolitcs from their UV, NMR, and mass spectral data, were not identified in incubations of C 3 H 1 0 T I / 2 cells with ACE. A C E - D N A adducts in C 3 H 1 0 T 1 / 2 cells were isolated, separated, identified, and quantitated using the 3~p-postlabeling method. A C E forms 4 major adducts and each was identified as an ACE-1,2-oxide/2'-deoxyguanosine adduct. The level of adduction was 2.18 pmoles ACE a d d u c t s / m g D N A after a 24-h incubation of ACE (16/xg/ml) with C 3 H | 0 T 1 / 2 cells. A C E - D N A adduct persistence and repair were evaluated in C 3 H 1 0 T 1 / 2 cells using a hydroxyurea block after A C E treatment. A C E - D N A adducts were not repaired under the conditions used in the morphologica_l transformation studies. Thus,

Correspondence: Dr. Stephen Nesnow, Carcinogenesis and Metabolism Branch, MD-68, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.A.). * This work was supported in part by the U.S. Environmental Protection Agency, Grant CR-811817, and Contract

68-02-4031, and USPHS Grant No. ES03433. A preliminary account of this work has been reported earlier (Sangaiah et al., 1985). * * N. Mohapatra is a National Academyof Science, National Research Council Resident Associateship Fellow.

0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

224

ACE provides an interesting example of a mutagenic PAH which is metabolized by C3H10T1/2 cells to active intermediates, forms relatively stable and persistent 2'-deoxyguanosine adducts in C3H10T1/2 cells, and yet induces no detectable morphological transforming activity under the experimental conditions used.

Aceanthrylene (ACE) is a cyclopenta-fused polycyclic aromatic hydrocarbon (CP-PAH) found in environmental samples (Katz et al., 1980) and combustion emissions (Spitzer and Dannecker, 1983) and results from the fusion of an ethene fragment to the periphery of anthracene (Fig. 1). Previously reported studies of ACE in Salmonella typhimurium strains TA98 and TA100 using an Aroclor-1254-induced rat river $9 suggested that ACE was highly active in inducing his ~ revertants (Kohan et al., 1985). The 5-membered ring in the CP-PAH provides a site for the metabolic activation of CP-PAH by the mammalian mixed-function oxidases. In 5 examples of other CP-PAHs studied, liver microsomes from Aroclor-1254-induced rats metabolized these CP-PAHs to cyclopenta-ring dihydrodiols (Gold and Eisenstadt, 1980; Nesnow et al., 1984) and using this metaboric activation system all the CP-PAH were mutagenic in Salmonella typhimurium (Eisenstadt and Gold, 1978; Nesnow et al., 1984) and were mutagenic in mammalian cells in culture (Gold et al., 1980; Kligerman et al., 1986; Nesnow et al., 1984). C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts have been used to study the metabolism and morphological transforming ability of PAH and several CP-PAHs (Gold et al., 1980; Mohapatra et al., 1987; Nesnow et al., 1981a; Rudo et al., 1986). The CP-PAH, cyclopenta[cd]pyrene and 3 of the 4 possible cyclopenta-benz[a]anthracene isomers were active in morphologically transforming C3H10T1/2 cells (Gold et al., 1980; Mohapatra et al., 1987). Cyclopenta[cd]pyrene and 2 of the 4 possible isomers of cyclopenta1

2

8

4 7

6

5

Fig. 1. Structure of ACE and its numbering system.

benz[a]anthracenes, benz[j]aceanthrylene, and bcnz[l]aceanthrylene, were metabolized by C3H10T1/2 cells to dihydrodiol metabolitcs including cyclopenta-ring dihydrodiols. This study examines the ability of ACE, the smallest CP-PAH yet examined, to be metabolized, to form DNA adducts, and to induce morphological transformation in C3HIOTI/2 mouse embryo fibroblasts. Materials and methods

Chemicals Synthesis and purification of ACE was performed according to published methods (Sangaiah et al., 1985). ACE-1,2-oxide was prepared by the method of McCaustland et al. (1980). The physicochemical and spectral data of this oxide were consistent with its structure and will be reported elsewhere. Trypsin (2.5%) and Dulbecco's PBS (KC1, 0.2 mg/ml; KH2PO4, 0.2 mg/ml; NaCI, 8 mg/ml; Na2HPO 4- 7H:O, 2.2 mg/ml) were obtained from Grand Island Biological Co. (Grand Island, NY). HPLC-grade ethyl acetate, methanol, and acetone were purchased from Burdick and Jackson Laboratories, Inc. (Muskegon, MI). Cell culture The mouse embryo fibroblast cell line C3H10T1/2 (passage 7), derived by Reznikoff et al. (1973a,b) was utilized in this study. Cell cultures were incubated in humidified incubators with an atmosphere 5% CO 2 in air at 37°C and 85% humidity. The cultures were grown in Eagle's basal medium with Earle's salts and L-glutamine supplemented with 10% heat-inactivated fetal bovine serum (Grand Island Biological Co.). The cells were checked on a routine basis for Mycoplasma contamination by Microbiological Associates, Bethesda, MD, by the Hoechst stain method and found to be Mycoplasma free. Morphological transformation assays The cell transformation and cytotoxicity procedures of Reznikoff et al. (1973b) (standard assay,

225 1-day protocol) and Nesnow et al. (1982) (delayed treatment assay, 5-day protocol) for C 3 H 1 0 T 1 / 2 cells were used without any modification. C 3 H 1 0 T 1 / 2 cells were seeded for transformation studies at 1000/dish (24 dishes/conc.) in 60-mm plastic petri dishes in 5 ml of medium. Cells were treated with ACE dissolved in acetone either l day or 5 days after seeding. After a 24-h exposure, the cells were fed with fresh complete medium containing 25 /~g/ml garamycin (Schering Corp., Kenilworth, N J) which has been shown to have minimal effects on the t r a n s f o r m a t i o n of C 3 H 1 0 T 1 / 2 cells (Bertram, 1979). 1 week after the treatment, the cytotoxicity dishes were fixed with methanol and stained with Giemsa. The media in the cell transformation dishes were changed weekly, and at confluence the fetal bovine serum was reduced to 5% (Bertram, 1977). At the end of 6 weeks, the dishes were fixed, stained, and scored for morphological transformation according to the criteria described by Reznikoff et al. (1973b). Metabolism studies ACE metabolites were identified from incubates of ACE with Aroclor-1254-induced rat liver microsomes (Nesnow et al., 1984). Aroclor-1254induced rat liver microsomes were incubated at 3 7 ° C for 15 min with agitation in a 5.0-ml incubation mixture which contained 50 m M phosphate buffer (pH 7.5), 5/~moles of N A D P + , 22.5 /~moles of glucose 6-phosphate, 9.0 units of glucose-6-phosphate dehydrogenase (Sigma Chemical Co., St. Louis, MO), 15 #moles of MgC12, and 300 nmoles of ACE dissolved in 250 /~1 of acetone. The incubation mixtures were extracted with 15 ml of ethyl acetate:acetone (2:1). 12 ml of the upper phase were removed and filtered through a 0.5-/~m Teflon filter (Miilipore Corp., Bedford, MA). The solvent was evaporated under a gentle stream of N 2 and stored at - 7 0 ° C. Metabolites were separated and collected using a Model 8800 high-pressure liquid chromatograph equipped with a 254-nm UV monitor (DuPont Instruments, Wilmington, DE). Each sample was reconstituted in 400 #1 of methanol for H P L C analysis and 50-/.tl aliquots were injected onto a DuPont Zorbax ODS reverse phase column (6.2 m m × 2 5 cm) and eluted at 2 m l / m i n with a

multistep gradient: Step 1: 65% aqueous methanol to 75% aqueous methanol over a 25-min time period using gradient No. 5; step 2: 75% aqueous methanol to 100% methanol over a 15-rain time period using gradient No. - 9. The retention times for the 3 A C E dihydrodiols were: ACE-1,2-dihydrodiol (17 min); ACE-9,10-dihydrodiol (19 rain); ACE-7,8-dihydrodiol (22 min). The UV absorbance was recorded, and the column effluent was collected in 0.5-ml fractions. (See Sangaiah et al., 1985; for a description of the H P L C chromatogram.) Metabolites were identified by UV, NMR, and mass spectrometry. The metabolism of ACE in homogenates of C 3 H I O T 1 / 2 cells was performed as follows. Logphase C 3 H 1 0 T 1 / 2 cells were grown in 75-cm 2 T-flasks in Basal Medium Eagle supplemented with glutamine and 10% fetal bovine serum. Upon approaching late log-phase growth the cells were trypsinized, washed thoroughly with Dulbecco's PBS, and freeze-thawed with liquid nitrogen. The cell homogenate was fortified with an N A D P H generator (see Metabolism studies) and incubated with ACE (16 lag/ml) in a final volume of 3 ml. After 30 min, the homogenate was extracted with ethyl acetate : acetone (2 : 1) and the extract chromatographed by HPLC. A 9.4 m m ID x 0.25 m Zorbax ODS column was used for these analyses with the previously described gradient. Under these conditions the elution times of ACE-1,2-dihydrodiol and ACE were 17.2 and 39.5 min respectively. Quantitation of the dihydrodiol concentrations was performed on a Hewlett Packard Model 3380A reporting integrator using anthracene as the external standard. Anthracene was selected as the external standard as it had a similar structure and molar extinction coefficient as ACE-1,2-dihydrodiol (20 800) and was available in sufficient quantity. DNA adducts Sixty 75-cm 2 flasks of C 3 H 1 0 T 1 / 2 cells which were approaching confluence were either treated with 16 /~g/ml ACE or 1 /~g/ml benzo[a]pyrene [B[a]P] for 24 h. The cells were washed either with Dulbecco's PBS (1 × ) or with complete medium (3 × ) . After the washing procedure, the flasks received complete medium and were then treated with 1 m M hydroxyurea and allowed to incubate

226 at 37°C for 0, 24 or 48 h (Lo and Kakunaga, 1982). At each time, 20 flasks were harvested (10 ACE and 10 B[a]P). Cells treated with acetone served as controls.

S9-Mediated modification of calf thymus DNA and polydeoxynucleotides with ACE Polydeoxyguanylic acid, polydeoxyadenylic acid, polythymidylic acid, and polydeoxycytidylic acid (Pharmacia-PL Biochemicals, Piscataway, N J) and calf thymus DNA (Sigma Chemical Co.) were dissolved in TE buffer (10 mM Tris-HC1, 1 mM Na2EDTA, pH 8.0) at 5 A260 units/ml. 50-/~g aliquots of calf thymus DNA or each polydeoxynucleotide were modified in a final volume of 2 ml containing 50 mM K2HPO4, 3 mM NazEDTA, 2 m g / m l Aroclor-1254-induced rat liver $9 protein, 1.8 units/ml glucose-6-phosphate dehydrogenase, 1 mM NADP +, and 4.5 mM glucose 6-phosphate. The reaction was initiated by adding 100/tl of 1.2 mM ACE dissolved in acetone and was incubated for 15 min at 37°C. The reaction was terminated by extracting the mixture 5 × with 1 vol. of diethyl ether. Residual ether was removed under a gentle stream of N 2 and the samples were stored at - 7 0 ° C until postlabeling analysis. Control samples containing the complete mixture without ACE or the complex mixture without $9 protein were prepared similarly.

Modification of calf thymus DNA with ACE-1,2oxide ACE-1,2-oxide was dissolved in DMSO at 1 mg/ml. A 100-/~1 aliquot of this solution was added to 100 #g of calf thymus DNA dissolved in 500 ~1 TE buffer and incubated at 3 7 ° C for 2 h. The samples were extracted and stored as described above. A control sample treated with DMSO alone was prepared similarly.

Isolation of DNA and analysis of adducts DNA was isolated from C 3 H 1 0 T 1 / 2 cells by a scaled-down version of a previously published solvent extraction procedure (Gupta, 1984), except that the homogenized cells were first digested with RNAases prior to digestion with proteinase K. Calf thymus DNA or polydeoxynucleotides were purified after incubation by removal of the endogenous RNA and protein by digestion with

RNAases and proteinase K and solvent extractions as above. DNA adducts were analyzed by 3ZP-postlabeling assay (Gupta et al., 1982) after enhancement of its detection limit using 1-butanol and tetrabutylammonium chloride to selectively enrich the mixture with ACE-Y-mononucleotide adducts prior to the labeling step (Gupta, 1985).

Results

Morphological transformation assays ACE was examined in the C 3 H 1 0 T l / 2 cell morphological transformation bioassay in which cells, 24 h after seeding, were treated for 24 h with ACE, allowed to grow to confluence, and scored for transformation 6 weeks later. ACE produced no type II or III loci in any of the 24 dishes treated and scored per concentration in 2 replicate studies at the following concentrations examined: 0.4, 0.8, 2.0, 4.0, 8.0, and 16.0 ~tg/ml (Table 1). The highest concentration of ACE used was 16 /zg/ml because of its insolubility in culture medium above that concentration. This observation is based on the persistent precipitation of ACE when administered at concentrations 20, 25, and 30 ~tg/ml. B[a]P used as the positive control produced an average of 1.8 type II and type III foci per dish in 96-100% of the treated dishes at a concentration of 1.0 /~g/ml. Spontaneous transformation was not detected in any of the 48 dishes treated with the solvent acetone. In our hands, spontaneous transformation of type II or type IIl loci is less than 0.005% (Nesnow et al., 1985b). ACE did not produce any significant cytotoxic effects at the concentrations tested. C 3 H 1 0 T 1 / 2 cells were treated with ACE using a protocol (delayed treatment protocol) which has been shown to be effective in enhancing the response of these cells to the transforming effects of chemicals (Nesnow et al., 1982). Dishes of cells were treated with ACE for 24 h on the fifth day after seeding. After 6 weeks of culture, the dishes were examined for morphological transformation. Two replicate studies were performed, each using concentrations of 0.4, 0.8, 2.0, 4.0, 8.0, and 16 # g / m l ACE and each with 24 dishes per concentration seeded and scored. One type II focus was observed at 2.0 ~ g / m l and one at 8.0 ~g/ml. This response was not considered significant (Ta-

227 TABI.E 1 SUMMARY: RESULTS FROM TREATMENT OF C 3 H 1 0 T 1 / 2 CELLS W I T H A C E U N D E R 2 T R E A T M E N T PROTOCOLS Treatment protocol

Total dishes with type II or Ill f o c i / total dishes scored

Standard protocol Seed cells, treat with A C E 24 h later, remove chemical after 24 h, and score for transformed loci after 6 weeks

0/288 a

Positive control B[ a IP (1.0/~ g / m l )

47/48

Negative control Acetone (0.5 %)

0/48

Delayed treatment protocol Seed cells, treat with A C E 120 h later, remove chemical 'after 24 h, and score for transformed foci after 6 weeks

2/288 a

Positive control B[a]P (1.0 btg/ml)

31/48

Negative control Acetone (0.5%)

0/48

a Data s u m m e d from all concentration levels tested.

ble 1). The concurrent positive control (B[a]P, 1.0 ~ g / m l ) gave an average of 1.1 type II and type llI loci/dish with 65% of the dishes exhibiting type II or II1 loci. The overall lower response observed with B[a]P in these 2 experiments compared to the previous studies is due to the use of a different lot of fetal bovine serum (Bertram, 1977). There were no type II or type III foci in any of the 48 dishes treated with acetone.

Identification of ACE metabolites from rat liver microsome incubations 3 major metabolites of ACE were identified and were used as standards for the ACE metabolism studies in C3HIOT1/2 cells. The metabolites were collected for characterization from semi-preparative HPLC. The elemental composition of each metabolite was determined by accurate mass measurement of the molecular ion and was consistent with a dihydrodiol formulation for all 3 metabo-

lites. The mass spectral fragmentation patterns of the 3 metabolites on electron impact were typical of dihydrodiol derivatives of PAH (Table 2). Detailed structures were established from the UV spectra and by analysis of the 1H-NMR spectra. The UV spectrum of the major metabolite was similar to that of anthracene and the 1H-NMR spectrum showed broadened singlets typical in appearance and chemical shift to the carbinyl protons of other cyclopenta-dihydrodiols (Table 2) (Nesnow et al., 1984). The absence both of vinyl resonances and of the AX quartet, with small coupling constants (5 Hz) characteristic of arene protons on an unsaturated cyclopenta-ring, confirms that the dihydrodiol functional~ation has occurred on the cyclopenta-ring rather than the anthracene nucleus. This metabolite was identical in both chromatographic and spectral properties to an authentic sample of trans-ACE-1,2-dihydrodiol (unpublished observation). By UV spectroscopy, the second metabolite (ACE-7,8-dihydrodiol) does not contain the anthracene chromophore (Table 2). The N M R spectrum revealed the presence of 2 vinyl resonances and the cyclopenta AX quartet requires that either the C 7 - C s or Cg-Ca0 bond of the benzo-ring forming the 'pseudo-bay region' be metabolized. The large downfield shift of one of the vinyl resonances (Ha0) is consistent with location of the vinyl bond in the pseudo-bay, and, on this basis, the 7,8-dihydrodiol structure has been assigned to the metabolite. The UV spectrum of the third metabolite (ACE-9,10-dihydrodiol) also suggests partial saturation of the anthracene nucleus (Table 2). The presence of 2 vinyl resonances and the cyclopenta AX quartet in the N M R therefore leads to the assignment of this metabolite as the complementary dihydrodiol, ACE-9,10-dihydrodiol. Consistent with the proposed structure, one of the carbinyl resonances (H10) appears highly deshieided, indicative of location of the dihydrodiol within the pseudo-bay. Additional support for the proposed structure is provided by the observation that the peri proton r e s o n a n c e ( H 6 ) of ACE-7,8-dihydrodiol does not appear downfield from the remaining aromatic resonances as it does for ACE-9,10dihydrodiol. A vinyl bond adjacent to a peri position, as in ACE-7,8-dihydrodiol, has been re-

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229 "FABLE 2 IDENTIFICATION OF ACE METABOLITES FROM RAT LIVER MICROSOMAL INCUBATIONS Metabolitc

UV "~ (2' .... )

Mass spectra b M / e (relative intensity)

NMR c

ACE-1,2-dihydrodiol

252 (major) 330, 348, 367, 383 (minor)

236 (100) [M] 218 (74) [M-It20 ] 190 (92) [M-H20, CO] 189 (87) [M-47]

8.49 (d, l i t ) Ilm; 8.43 (S, 1H) H6; 8.15 (d, 1H, J = 7.6 Hz, and 7.92 (d, 1ti, J = 5.8 Hz) H 5, H7; 7.68-7.45 (m, 4H) H 3, H4, H8, Hg; 5.88 (br.s, 1H) H1; 5.52 (br.s, 1H) H2; 5.03 (br.s, 1H) and 4.93 (br.s, 1 H) OH

ACE-7,8-dihydrodiol

247, 254 (major) 294sh, 307(sh), 333, 347, 365 (minor)

236 (100) [M] 218 (76) [M-H201 190 (93) [M-H20, CO] 189 (87) [M-471

7.74 (d, 1H, J4.~ = 6.3 Hz) Hs; 7.65 (s, IH) H6; 7.68-7.50 (m, 2H) H 3, H 4 7.55 (br.s, 1H) H1; 7.07 (br.s, 1H) H2; 6.69 (br., unresolved, 1 H) H7; 6.05 (br., unresolved, 1H) Hs; 5.11 (br.s, 1H); Hao 4.52 (br.s, 1H) Hg; 4.84 (br.s, 1tt) and 4.80 (br.s, IH) OH

ACE-9,10-dihydrodiol

253, 270(sh) (major) 314, 327, 339, 358 (minor)

236 (100) [M] 218 (75) [M-H20 ] 190 (100) [M-H20 , CO]

8.05 (s, 1H) He; 7.79 (d, IH, Js.4 = 8.3 llz) Hs; 7.68 (d, 1H, J3,4 = 7.3 Hz) H3; 7.53 (m, 1H) H4; 7.27 (d, 1H, JL2 = 4.2 Hz) and 7.09 (d, 1H, JL2 = 4.2 Hz) H1, H2; 7.02 (d, IH, Jr0.9 = 10.4 Hz) Hm; 6.25 (d, 1H, Jg.m = 10.4 Hz) H9; 4.86, 4.81 (AB quartet, 2H, J = 7.3 Hz) H7, H8; 4.48 (br.s, 1H) and 4.43 (br.s, 1H) OH

189 (80) [M-471

a UV spectra determined in methanol. b Electron impact mass spectra at 70 eV. NMR spectra (250 MHz, acetone-d6).

ported to cause a downfield shift of the peri proton resonance in structurally related analogs (Fu and Harvey, 1977).

Metabolism of ACE by C3HIOT1/2 cells Determination of the metabolism of ACE was performed using a cell homogenate prepared from

washed log-phase C 3 H 1 0 T 1 / 2 cells and supplemented with N A D P H . Organic extracts of the incubation mixture were subjected to reverse-phase HPLC. The only peak observed in the H P L C trace at 254 nm in addition to unmetabolized ACE was identified as ACE-l,2-dihydrodiol using microsomal A C E metabolite standards. The mean

Fig. 2. 32p-Postlabeling analysis of ACE-DNA adducts. 32p-Adduct maps of calf thymus DNA (DNA), DNA from C3H10T1/2 cells (T1/2 cells) or poly dGuo exposed to ACE, ACE-1,2-oxide, B[a]P, or acetone solvent (sham), in the presence or absence of $9, as indicated. After enzymatic hydrolysis of DNA (5 #g), adducts were enriched by extraction into l-butanol, 5'-32p-labeled with carrier-free [~-32p]ATP ( >~3000 Ci/mmole) and chromatographed by multidircctional PEI cellulose TLC in the solvents previously described (Gupta, 1985) except that D4 solvent in g-i was isopropanol : 4N ammonia, 1 : 1. Spots were detected by screen-enhanced autoradiography at - 8 0 ° C for 4-12 h. Spots encircled were only detectable after prolonged exposure. (a) C3H10TI/2 cells exposed to acetone for 24 h; (b) C3H10T1/2 cells exposed to ACE for 24 h; (c) C3H10T1/2 cells exposed to B[a]P for 24 h. Adduct No. 8 is BPDEI-dGuo; (d) calf thymus DNA modified with ACE in the presence of an Aroclor-1254-induced rat liver $9; (e) poly dGuo modified with ACE in the presence of an Aroclor-1254-induced rat liver $9; (f) cochromatography of materials described in d and e; (g) C3H10T1/2 cells exposed to ACE for 24 h; (h) calf thymus I)NA exposed to ACE-1,2-oxide; (i) cochromatography of materials described in g and h.

230 metabolic rate of formation (_+standard deviation) of this cyclopenta-ring dihydrodiol in triplicate studies was 450 _+ 50 p m o l e s / h / 1 0 6 cells and was similar to that previously reported for total B[a]P metabolism in C 3 H 1 0 T 1 / 2 cells (Nesnow et al., 1981a).

ACE-DNA adducts in C3HIOT1/2 cells A C E - D N A adducts were analyzed and identified by the 32p-postlabeling technique of Gupta (1985). The butanol extraction modification of the 32p-postlabeling technique was used for these studies as it has been previously shown that many P A H - D N A adducts are selectively enriched by this procedure ( G u p t a and Earley, 1988). C 3 H 1 0 T I / 2 cells treated v,4th ACE for 24 h formed 5 adducts, 4 were major adducts (Nos. 2-5) (Fig. 2b), while no adducts were observed in the DNA of C3H10T1/2 cells treated with acetone (Fig. 2a). In order to identify the DNA base portion of the adducts, ACE was incubated with an Aroclor-1254-induced rat liver $9 and calf thymus DNA (Fig. 2d). 5 adducts were also observed, 4 corresponding to the major adducts found in the DNA of C3H10T1/2 cells treated with ACE (Nos. 2, 3, 4, 5) and adduct (No. 6) not present among the adducts in C 3 H 1 0 T 1 / 2 cells. The autoradiograms of adducts from the modification of individual homopolymers of 2'-deoxyguanosine (dGuo), 2'-deoxyadenosine (not shown), 2'-deoxycytidine (not shown), and thymidine (not shown) by incubation with Aroclor-1254-induced rat liver $9 and ACE strongly suggested that adducts Nos. 2, 3, 4, 5 were d G u o adducts (Fig. 2e) and co-chromatography of the mixture confirmed this conclusion (Fig. 2f). As a control, B [ a ] P - D N A adducts in C 3 H 1 0 T 1 / 2 cells exposed to 1/~g/ml B[a]P for 24 h formed several adducts, with adduct No. 8 (anti-trans-B[a]P-7,8dihydrodiol-9,10-oxide-dGuo (BPDEI-dGuo]) representing 88-90% of the total adducts with an adduction level of 38.5-50 pmoles/mg DNA (Fig. 2c). The position of attachment of each adduct with respect to ACE was examined by incubating ACE1,2-oxide with calf thymus DNA and comparing the autoradiograms with those obtained with ACE-treated C3HIOT1/2 cells and using a different D4 solvent mixture than that used in panel

a-f. Individual chromatograms and co-chromatograms (Fig. 2g-i) indicated that adduct Nos. 2, 3, 4, and 5 were ACE-1,2-oxide adducts. Therefore, the 4 major ACE adducts in C 3 H 1 0 T 1 / 2 cells are ACE-1,2-oxide-dGuo adducts. Adduction levels of ACE in C 3 H I O T I / 2 cells exposed for 24 h to 16 ~ g / m l ACE were 2.18 pmoles ACE a d d u c t s / m g DNA from triplicate determinations with the predominant adduct No. 5 representing 38%, adduct Nos. 2, 3, 4 representing 20% each and adduct No. 1 representing 2% of the total.

Persistence and repair of ACE adducts in C3HIOTI / 2 cells The repair of A C E - D N A adducts in C 3 H 1 0 T 1 / 2 cells was examined by treating logphase cultures of cells with ACE (16 ~ g / m l ) or B[a]P (1 /~g/ml), removing the PAH after 24 h and adding 1 mM hydroxyurea to inhibit DNA synthesis. Cells were examined for A C E - D N A adducts or B [ a ] P - D N A adducts 0, 24, and 48 h after the addition of hydroxyurea. Under the normal conditions used to remove chemicals from dishes of C 3 H 1 0 T I / 2 cells using a single rinsing procedure with PBS both A C E - and B [ a ] P - D N A adducts increased 50-73%, 48 h after the hydroxyurea block (Fig. 3). This was presumably due to the persistence of the PAH absorbed to the plastic petri dishes. However, under more stringent rinsing conditions with complete medium where 40% of the B [ a ] P - D N A adducts were repaired by 48 h, no repair was observed in the DNA of ACE-treated cells. Again a 75% increase in binding was noted. The increase in A C E - D N A adducts after thorough washing may be due to a depot storage form of ACE within C 3 H 1 0 T 1 / 2 cells. The proportion of A C E - D N A adducts did not change appreciably with incubation time in either rinsing protocol (data not shown). Discussion

In previous studies it has been shown that CP-PAHs are more active as genotoxins and mouse skin tumor initiators than the parent PAH from which they are derived. A case in point is cyclopenta[cd]pyrenc which morphologically transforms mammalian cells and is a mouse skin tumor

231 200

t001 ~

" ""

B(a)P-einole wa B(a)P-'e2ngle waeh

75

5O

24 ,48 Tilde AFTERHYDROXYUREABLOCK, HR Fig. 3. Persistence and repair of A C E and B [ a I P - D N A adducts in C 3 H 1 0 T 1 / 2 cells. Cells were treated with A C E or B[a]P for 24 h. The dishes were rinsed once with PBS (single wash) or rinsed 3 × with complete medium (multiwash) and 1 m M hydroxyurea added. Dishes were harvested 0, 24, and 48 h later. Total levels of D N A adducts in p m o l e s / m g D N A in cells harvested at 0 h were: ACE-single wash, 2.14; ACE-multiwash, 2.2; B[a]P-single wash, 42.9; B[a]P-multiwash, 56.2. The error bars represent the standard errors from duplicate determinations. 0

initiator in CD-1 and Sencar mice (Cavalieri et al., 1981; Gold et al., 1980; Raveh et al., 1982). Pyrene, the parent PAH of cyclopenda[ed]pyrene, is inactive in these bioassays (IARC, 1983a; Nesnow et al., 1987). Similarly, anthracene (the parent PAH of ACE) is inactive as a gene mutagen in bacterial and manunalian cells, is inactive in morphologically transforming mammalian cells and is inactive as a mouse skin tumor initiator (IARC, 1983b; Nesnow et al., 1987). Both pyrene and anthracene are also inactive in forming DNA adducts in human peripheral lymphocytes (Gupta et al., 1988). We have shown that ACE is a potent gene mutagen in Salmonella typhimuriurn (Kohan et al., 1985) and by analogy to the behavior of cyciopenta[cd]pyrene, benz[j]aceanthrylene, benz[l]aceanthrylene, and benz[e]aceanthrylene we, therefore, expected to observe morphological transformation of C 3 H 1 0 T 1 / 2 cells in culture. However, ACE was inactive in these cells. The lack of of solubility of ACE may be a limiting factor in the evaluation of this CP-PAH in C 3 H I O T 1 / 2 cells. However, this insolubility is not unusual as other PAHs and CP-PAHs cannot be evaluated at concentrations above 20 /~g/ml. The lack of cytotoxicity of ACE which might be initially considered as evidence of lack of suffi-

cient exposure concentration is also a general phenomenon observed with CP-PAH which can transform C 3 H I O T 1 / 2 cells such as 3-methylcholanthrene, cyclopenta[cd]pyrene (Gold et al., 1980; Reznikoff et al., 1973b) and the 3 cyclopenta-fused isomers of benz[a]anthracene (Mohapatra et al., 1987). Therefore, under maximal bioassay conditions ACE has no observable morphological transforming activity given the sensitivity of the bioassay system. Theoretical considerations also support the hypothesis of genotoxicity for ACE. The value of the AEd~k,Jfl for the carbonium ion derived from ring opening of ACE-1,2-oxide is 0.931, considerably larger than that value for BPDEI (0.794) (Sangaiah et al., 1985). This suggests that ACE should be a powerful genotoxic agent if metabolized to the 1,2-oxide. The inability of ACE to transform these cells under 2 different treatment protocols could be related to deficiencies in metabolism, DNA adduction, or to differences in the repair or persistence of the A C E - D N A adducts. Each of these possibilities was examined in studies in C 3 H 1 0 T 1 / 2 target cells. ACE is metabolized by C 3 H 1 0 T I / 2 cells to ACE-l,2-dihydrodiol with a rate of formation that is quite high compared to the metabolism at the cyclopenta-ring of other CP-PAHs: cyclopenta[cd ]pyrene, benz[ j]aceanthrylene, benz[ l]aceanthrylene or when compared to metabolism of B[a]P at the 7,8-bond (on the metabolic pathway to BPDE) (Table 3). The differences in these rates can be as much as 15-fold between ACE and the other CP-PAHs studied. The detection of ACE1,2-dihydrodiol as the metabolic product in C 3 H 1 0 T 1 / 2 cells is strong evidence for the formation of the metabolic precursor, ACE-1,2-oxide. This oxide has been shown to mutate Salmonella typhimurium (unpublished data) and other CPPAH oxides (at the cyclopenta-ring) such as cyclopenta[cd]pyrene-3,4-oxide and benz[j]aceanthrylene-l,2-oxide, are effective at mutating Salmonella typhimurium and morphologically transforming C 3 H I O T 1 / 2 cells (Bartzcak et al., 1987; Eisenstadt and Gold, 1978; Gold et al., 1980; Nesnow, unpublished results). ACE forms 4 major stable DNA adducts in C 3 H 1 0 T 1 / 2 cells which have been identified as d G u o adducts with ACE-l,2-oxide. The semi-em-

232 TABLE 3 C O M P A R I S O N OF T H E METABOLISM, D N A A D D U C T FORMATION AND MORPHOLOGICAL TRANSFORM I N G ACTIVITY OF PAH IN C 3 H 1 0 T 1 / 2 CELLS PAH

ACE

Total metabolism rate (pmoles/h/ 10 6 cells) a

Total DNA adducts (pmoles/ mg DNA) b

450 (450) 2.18 +_50 (n = 3) Cyclopenta- 52 (38) 7.0 ~ [cdlpyrene -_,3 (n = 3) o Benz[j]ace215 (30.9) 0.32 e anth~'lene _+69 (n = 6) g Benz[ I ]ace623 (32.9) 1.54 ¢ anthrylene 5:161 (n = 6) ~ B[a]P 287 (187) 43-56 _+84 (n = 5) h

Morphological transformation (% dishes with type II/111 foci) ~

0 (16/tg/ml) 100 (10/tg/ml) f 100 (2.5 ~tg/ml) g 85 (10 ,ttg/ml) g 92 (1.0 p.g/ml) g

a Mean + SD for n replicate studies. Value in parentheses is metabolite rate at the cyclopenta-ring (7,8-dioi for B[alP). b D N A adduct measurements by the method of Gupta (1985). Value in parentheses is the concentration which gives the above responses. d Nesnow et al. (1981b). e C 3 H 1 0 T 1 / 2 cells were incubated with each P A H (1.0 p.g/ml) for 24 h (unpublished results). e Gold et al. (1980). Mohapatra et al. (1987). h Nesnow et al. (1981a).

pirical and ab initio calculations of the A EoeloJfl of the ring-opened carbocation of ACE-1,2-oxide suggests that initial attack of a nucleophile would be on position 1 (Rabinowitz et al., unpublished; Sangaiah et al., 1985). Possible structures of some of the A C E - D N A adducts could be isomers of N 2-(l-(1,2-dihydro-2-hydroxy-aceanthrylenyl))-

2"-deoxyguanosine, and O6-(1-(1,2-dihydro-2-hydroxy-aceanth rylenyl))-2'-deoxyguanosine. Thc number of adducts may reflect a combination of effects including adduction at multiple sites on dGuo, stereo and geometric isomers, as wcll as some instability of the initially formed adducts lcading to further degradation such as dehydration or oxidation. The observation of dGuo adducts of PAH is commonly associated with the mutagenic and carcinogenic activity of

these chemicals and formation of A C E - d G u o adducts is therefore consistent with the expectation of biological activity (Hemminki, 1983; Singer and Grunberger, 1983). The level of adduction observed with ACE in C 3 H 1 0 T 1 / 2 cells, although not as high as that observed with B[a]P, is consistent with the level of adduction with other CP-PAHs known to transform these cells (Table 3). The appearance of a DNA adduct (adduct No. 6) in calf thymus DNA after incubation with ACE and an Aroclor-1254-induced rat liver $9 which is not present in the DNA of C3HIOT1/2 cells exposed to ACE suggests a divergent pathway in the 2 experimental systems. At this time we have little direct evidence linking specific adducts with mutagenic or cell-transforming events. Whether this divergent pathway is involved in the mutagenie activity of ACE rcmains to be explored. However, the weight of evidence suggests that since the major Aroclor-1254-induced rat liver $9 metabolite was ACE-1,2-dihydrodiol, the major active metabolic intermediate generated in the Salmonella tpphimurium assays would therefore be ACE-1,2-oxide. Since ACE-l,2-oxide is mutagenic in Salmonella typhimurium, it would seem reasonable that the major A C E - D N A adducts contributing to mutagenesis in Salmonella typhimurium would be ACE-1,2-oxide-derived adducts. These are the same adducts as found in ACE-exposed C 3 H 1 0 T I / 2 cells. Therefore, the weight of circumstantial evidence leatJs to the expectation of transforming activity of ACE in C 3 H I O T 1 / 2 cells. Rapid repair of A C E - D N A adducts might have provided an explanation for the resistance of C 3 H 1 0 T 1 / 2 cells towards ACE but is ruled out by the observation that A C E - D N A adducts persist up to 48 h after treatment under the conditions used to assay transformation in these cells. Brown ct al. (1979) have reported a slow repair process in these ceils after treatment with B[a]P, a phenomenon also observed for this compound in our experiments. Several classes of explanation can be proposed to explain these results: those based on the nature of the C 3 H 1 0 T I / 2 cell transformation system, and those based on the formation of site-specific DNA adducts with regard to effects on regulatory genes.

233 One explanation for these observations is that ACE could be a ' p u r e ' initiator of morphological transformation in C 3 H I 0 T 1 / 2 cells and requires the action of a tumor promotor to express its effects. For example, treatment of C 3 H 1 0 T 1 / 2 cells with N-methyl-N-nitro-N'-nitrosoguanidine 24 h after seeding does not transform them (Reznikoff et al., 1973b), but if these exposed cells are subsequently treated repetitively with 12-O-tetradecanoylphorboi-13-acetate, morphologically transformed loci appear (Frazelle et al., 1984). However, it has also been found that when C 3 H 1 0 T 1 / 2 cells are treated with N-methyl-Nnitro-N'-nitrosoguanidine in a delayed treatment protocol (5 days after seeding), morphologically transformed loci will be produced without the need for tumor promoter treatment (Nesnow et al., 1982). Similar results have been observed with propane sultone and aflatoxin B~ (Nesnow et al., 1982). Enhanced morphological transformation has been observed by comparing the standard and delayed treatment protocol with PAHs such as B[a]P, 7,12-dimethylbenz[a]anthracene and 3methylcholanthrene (Nesnow et al., 1985a). In the present studies, A C E did not t r a n s f o r m C 3 H 1 0 T 1 / 2 cells when applied in the normal (1-day) or the delayed (5-day) protocol. Without data on the performance of ACE in a 2-stage morphological transformation system, a conclusion regarding ACE as a ' p u r e ' initiator of morphological transformation cannot be made. Therefore, if ACE does possess the ability to transform these cells, its activity is below the level detectable by the delayed treatment protocol. C 3 H 1 0 T 1 / 2 cells transformed by 3-methylcholanthrene have been shown to exhibit an altered c-Ki-ras homologue (Parada and Weinberg, 1983), and elevated expression of c-myc has been reported in cells transformed with 7,12-dimethylbenz[a]anthracene and bleomycin (Billings et al., 1987). The involvement of altered oncogenes in cellular transformation has also been demonstrated by induction of foci by transfection with v-mos and c-Ha-ras (Van der Hoorn and Muller, 1985; Manoharan ct al., 1985). If the induction of specific activating mutations in cellular protooncogenes represents a critical step in the induction of loci in C 3 H I O T 1 / 2 cells, then it is possible that the lack of observed focus induction in this

study results from either the inability of ACE to produce adducts at these specific D N A sequences, or the failure of adducts to induce the proper activating mutation. The sequence specificity of D N A adduction and mutation induction by ACE is presently unknown, but other PAHs have been demonstrated to induce specific mutations nonrandomly in D N A (Eisenstadt et al., 1982). In conclusion, the observation of significant formation of A C E - D N A d G u o adducts in transformable mouse embryo fibroblasts without concomitant morphological transformation provides a good model system for the study of the cancer process on a molecular level and specifically provides a method to study the relationships between chemical structure and biological activity of PAH and CP-PAH.

Acknowledgements We wish to thank Miss Joye Denning and Mrs. Shirley Milton for manuscript preparation. The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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