Mutation induction and DNA adduct formation in Chinese hamster ovary cells treated with 6-nitrochrysene, 6-aminochrysene and their metabolites

MurcriiorzResemci~. ‘79 ( I YY3 153- 16-t $1 !YY2 Elscvier Science Publishers R.V. All rights reserved Ol6S-l2lH/Y2/SOS.OO

MUTGEN

01760

Mutation induction and DNA adduct formation in Chinese hamster ovary cells treated with 6-nitrochrysene, 6-aminochrysene and their metabolites K. Barry

Delclos

and Robert

H. Heflich

Food and Drug Adtninistratiort. Nutionul Center for Toxicologicul Research, Jefferson. AR 72079 (U.S.A.) (Received

I I March 1991)

(Revision received 3 October (Accepted 2X October

Keywords: Chinese hamster ovary cells: DNA

adducts; h-Nitrochrysene:

1991)

199 1J

6-Aminochrysene;

Mutation

induction

Summary

6-Nitrochrysene, 6-aminochrysene and several of their metabolites were assayed for mutagenic activity at the hypoxanthine-guanine phosphoribosyl transferase (hprt ) locus in DNA-repair-proficient Chinese hamster ovary (CHO-Kl 1 cells and excision-repair-deficient CHO-UVS cells. Mutagen-DNA adducts were analyzed by “‘P-postlabeling in cells treated under the conditions of the mutagenicity assay and compared with the adduct patterns produced from the in vitro reaction of metabolites of 6-nitrochrysene and 6-aminochrysene with calf-thymus DNA. The mutagenic activities of the test compounds in the presence of a liver homogenate (S9) fraction from Aroclor 1254-pretreated rats, expressed as the number of mutants per 10h cells per nmole test compound per ml, in CHO-Kl and CHO-UVS cells, respectively, were as follows: 6nitrochrysene, 0.3 and 4; 6-aminochrysene, 35 and 117; 6-nitrochrysene-1,2_dihydrodiol, 1 and 6; 6-aminochrysene- 1,2-dihydrodiol, 488 and 644; chrysene (run as a positive control), 12 and 28. 6-Nitrosochrysene was a direct-acting mutagen, yielding 127 and 618 mutants per lOh cells per nmole per ml in CHO-Kl and CHO-UV5 cells, respectively. Mutagen-DNA adduct analysis indicated that cells treated with 6aminochrysene in the presence of S9 or 6nitrosochrysene in the absence of S9 contained an adduct pattern identical to that derived from the in vitro reaction of N-hydroxyd-aminochrysene with calf-thymus DNA. Cells treated with 6-aminochrysene-1,2-dihydrodiol plus S9 contained a single mutagen-DNA adduct that was distinct from those derived from N-hydroxy-6-aminochrysene. Based on comparison with previous studies, this adduct is presumed to be derived from 1,2-dihydroxy-3,4-epoxy-1,2,3,4_tetrahydro6-aminochrysene. Cells treated with 6-nitrochrysene plus S9 and 6-nitrochrysene-1,2_dihydrodiol plus S9 contained a single major chromatographically identical adduct that was apparently derived from N-hydroxy-6-aminochrysene-1,Zdihydrodiol. The results indicate that 6-nitrochrysene. 6-aminochrysene and their metabolites are mutagenic in CHO cells, but that the major activation pathway for 6nitrochrysene and 6-nitrochrysene-1,Zdihydrodiol in this system differs from previously described pathways.

Abbrcriurions: 6-AC,

6-aminochrysene;

6-AC-1.2-dihydrodiol.

1,2-dihydro-1,2-dihydroxy-6-aminochrysene; Correspondence:

Dr. K. Barry Delclos. Division of Biochemi-

cat Toxicology, HFT-I IO, National Center Research, Jefferson, AR 72079 (U.S.A.).

for Toxicological

hamster ovary; DMSO.

dimethyl

CHO,

sulfoxide; MN,

Chinese

micrococcal

nuclease; 6-NC. h-nitrochrysene; 6-NC-I,?-dihydrodiol, hydro-1.2-dihydroxy-6nitrochrysene;

NitroPAH,

nitro

1,2-dipoly-

cyclic aromatic hydrocarbon; SPD, spleen phosphodiesterase.

The well-documented mutagenicity and tun1origenicit.y of many nitro polycyclic aromatic hydrocarbons (nitro PAH) in experimental models have raised the issue of the potential hazards of human exposure to these compounds and have stimulated a considerable amount of work on their metabolism (reviewed in Fu, 1990; Rosenkranz and Mermelstein. 1985; Tokiwa and Ohnishi, 1986). Nitro PAHs can be metabolized in vivo by nitroreduction to yield nitroso, N-hydroxy and amino derivatives. or by ring oxidation to yield epoxides. phenols, dihydrodiols and diol epoxides analogous to those formed by the metabolism of polycyclic aromatic hydrocarbons (Fu. 1990). While several nitro PAHs have become objects of study due to their quantitative importance in the environment or their mutagenic activities in Salmonella tester strains, 6nitrochtysene (6-NC). an air pollutant and a component of diesel exhaust, came to be of interest primarily because of its carcinogenic activity in the preweanling mouse bioassay (Busby et al., 1985, 1988, 1989; El-Bayoumy et al., 1989a; Wislocki, 1986). 6Aminochrysene (6-AC) has also been shown to bc tumorigenic in mice (ElBayoumy et al., 1989a; Lambelin et al.. 1975; Roe et al., 1969) and has been tested as an antitumor agent in humans (Groupe Europeen du Cancer du Sein. 1967: Sculier et al., 1989). While there are multiple potential activation pathways for h-NC and 6-AC. previous in vitro and in vivo studies have shown that both 6-NC and 6-AC are metabolized to reactive electrophilic intermediates primarily through the intermediate formation of N-hydroxy-6-AC or 6AC-I.?-dihydrodiol (Delclos et al., 1987a. b, 1988, 1959, 1990; El-Bayoumy et al., 1989b). This latter pathway is the major activation pathway for 6-NC under conditions where its potency as a moLrsc lung and liver carcinogen has been demonstrated (Delclos et al., 1987b, 1988). The preferred pathway of metabolic activation for 6-NC is apparently determined by the xenobiotic metabolizing enzymes present in the test system and is thus dependent on such factors as the target organ (Del&s et al., 1990), age of the animal (Delclos et al., 1987b, 1990) and prior exposure to enzyme inducers (Delclos et al., 1989). The activation of 6-K by ring oxidation pathways contrasts with

the general finding that the activation of aromatic amines involves the obligatory formation of Noxidized metabolites. In addition, when activation of 6-AC through N-oxidation does occur, it differs from that of the majority of aromatic amines that have been studied. First, indirect evidence (Lubet et al., 1989) suggests that, like 4,4’-methylene-bis(Zchloroaniline) but unlike other aromatic amines that have been examined (Butler et al., 1989), 6-AC is preferentially N-oxidized by phenobarbital-inducible form(s) of cytochromes P-450. Second, with few exceptions, the major carcinogen-DNA adducts derived from Noxidized aromatic amines and their ester derivatives are formed through reaction at the C8 position of deoxyguanosine (Beland and Kadlubar, 1990). While the activation of 6-NC or 6-AC through the formation of IV-hydroxy-6-AC does generate a C8deoxyguanosine adduct, C8-modified deoxyinosine, presumably derived from oxidation of the corresponding C8-modified deoxyadenosine derivative, and N2-modified deoxyguanosine have also been identified as major products (Delclos et al., 1987a. 1989, 1990; ElBayoumy et al., 1989b). We have been interested in evaluating the roles of the various metabolites of 6-NC and 6-AC and the carcinogen-DNA adducts formed from these metabolites in determining the biological activities of these compounds. In the present study, we have examined the mutagenicities of 6-NC and its metabolites (Fig. 1) in DNA excision-repair-proficient and repair-deficient Chinese hamster ovary (CHO)

AHI h-Aminorhryscnr

Lo,

Fig.

4%

h-Ni~rurhrysmr

6-Aminochrywne

I .Z-Dihydrodiol

I .2-Dihydrodiol

I. Structures

of h-nitrochrysene.

6-aminochrysene

and

their derivatives that were tested for mutagenic activity in this study.

cells and have determined the DNA adduct profiles associated with the mutagen treatments. The purpose of these experiments was to establish the mutagenic activity of 6-NC and its metabolites under the standard conditions of the CHO mutagenesis assay and to establish the mutagen-DNA adduct spectra associated with the treatments. This information will be useful for the selection of treatment conditions for analyses of mutation spectra induced by mutagen--DNA adducts resulting from specific metabolic activation pathways of 6-NC and 6-AC. Materials and methods Mutagens and other chemicals

6-NC and chrysene were obtained from Chemsyn Science Labo:atories, Lenexa, KS. 6-AC was purchased from Aldrich Chemical Co., Milwaukee, WI and was lcrther purified prior to use by open column silica gel chromatography with toluene and toluene/methanol as eluting solvents. 6-Nitrosochrysene and N-hydroxy-6aminochrysene (Delclos et al., 1987a), 6-NC- 1,2dihydrodiol (El-Bayoumy and Hecht, 1984) and 6-AC-1,Zdihydrodiol (Delclos et al., 1988) were prepared as described in the indicated references. DNAase I, micrococcal nuclease, nuclease Pl, and spleen and snake venom phosphodiesterases were from Sigma Chemical Co., St. Louis, MO. T4 Polynucleotide kinase was from United States Biochemical Corporation, Cleveland, OH. [y- ‘*P]ATP was synthesized from carrier-free [ ‘* PIphosphate (ICN Biomedicals, Costa Mesa, CA) as described by Gupta et al. (19821. Mutagenicity assays

Mutations at the hprt locus were measured in DNA-repair-proficient CHO-Kl cells (strain CHO-Kl-BH,, obtained from A.W. Hsie, University of Texas, Galveston, TX) and DNA-excision-repair-deficient CHO-UVS cells (obtained from L.H. Thompson, Lawrence Livermore National Laboratory, Livermore, CA) by assaying resistance to 10 PM 6-thioguanine. Assays were conducted in the presence and absence of liver S9 (final concentration 400 pg protein per ml) from Aroclor 1254-pretreated rats using the general procedures of Machanoff et al. (19811, modi-

ficd as follows. Cultures, initiated at a conccntration of 1 X lOh cells/lOO-mm petri dish on the preceeding day, were exposed in duplicate for 5 h at 37°C to dimethyl sulfoxide (DMSO) or test chemicals dissolved in DMSO (final DMSO concentration in control and test plates was 1% 1, washed with calcium-magnesium-free phosphatebuffered saline and fed with nutrient mixture F-12 containing 5% fetal bovine serum. Cytotoxicity was determined by measuring relative cloning ability on the next day, and mutations were quantitated after 8 days of phenotypic expression. A minimum of 2 x 10” cells were maintained during the phenotypic expression period and 2 x 10“ cells from each exposure group were tested for 6thioguanine resistance. A benzo[a]pyrene (5 pg/ml for CHO-Kl cells, 0.75 pg/ml for CHOUV5 cells) treatment group was included as a positive control in all assays with S9. In most cases, assays with and without S9 were conducted simultaneously so no specific positive control without S9 was performed. DNA adduct analysis

CHO-Kl or CHO-UV5 cells were treated with the concentrations of test compounds indicated in the text using the procedures outlined for the mutation assay. DNA was %oiated from the CHO cells (2-7 X 10’ cells) by standard methods (Beland et al., 1984) and was dissolved in 5 mM bis-tris, 0.1 mM EDTA. pH 7.1. Calf-thymus DNA modified by N-hydroxy-6-AC or metabolites of 6-NC- 1,2-dihydrodiol or 6-AC-1,2-dihydrodiol was prepared as previously described (Delclos et al., 1987a, 19881. DNA (2 pg) was hydrolyzed to nucleoside 3’-monophosphates by incubating with micrococcal nuclease (0.012 U,/pg) and spleen phosphodiesterase (0.001 U/pg) in 20 mM sodium succinate, 10 mM CaCl,, pH 6.0, at 37°C for 3.5 h (Gupta et al., 1982). The DNA digest was then treated with nuclease Pl (0.54 U/pug DNA) for 1 h at 37°C (Reddy and Randerath, 1986) or extracted with n-butanol (Gupta, 19851, [5’-“Plphosphorylated at 37°C for 40 min with T4 polynucleotide kinase (1 U/pg DNA) and 200 PCi [$*P]ATP (Gupta et al., 1982) and analyzed chromatographically by a contact-transfer method (Lu et al., 1986). The !Ibutanol extraction procedure used differed from

that ci~ribed by Gupta hutsno] exttracts were not

(1985) in that the IIback-washed with ~ater. This change in the procedure was based on espriments with micrococcal nuclease/ spleen phosphodiesterase (MN/SPD) digests of [‘HINhydroky-6-AC-modified DNA that indicated up to jO% of the radioactivity in the n-butanol extracts was removed during the back-washing step. “P-Labeled mutagen-nucleotide adducts were separated from unmodified nucleotides (Dl) using Machery-Nagel PEI cellulose plates from Alltech Associates. Inc., Deerfield, IL with 0.6s M sodium phosphate buffer. pH 6.0, as the developing solvent. Higher D1 buffer concentrations were found to move the adduct derived from 6-AC-1,2-dihydrodiol off the origin (G. Talaska, unpublished data) and were therefore not used. The adducts, which remained as a compact spot at the origin of the Dl plate, were excised and placed in contact with the origin (1.5 cm from bottom, 1.5 cm from left border) of a 10 X 10 cm E. Merck PEI cellulose plate (Alltech Associates, Inc.) for transfer and development in the D3 (3.3 M lithium formate, 7.7 M urea, pH 8.2) and D4 (0.72 M sodium phosphate, 0.45 M Tris, 7.6 M urea, pH 8.2) directions. Following development in the D4 direction, a wick (1.2 cm in width) was attached to the top of the plate (top with respect to D4 development direction) and plates were washed with 0.9 M sodium phosphate buffer, pH 6.8, until the solvent reached the top of the wick. The wicks were removed and the corners of the plate marked with radioactive ink spots. Adduct spots were visualized by placing the plates in contact with Cronex X-r&y fitm ~DuPont, Wilmington, DE) for a length of time determined by scanning the plate with a survey meter equipped with a Geiger-Mueller detector.

Calf-thymus DNA was dissolved at 2 mg/ml in 20 mM sodium phosphate, pH 7.0. 20 ~1 of a 50 pg/ml solution of 6-nitrosochrysene in DMSO were added to aliquots of the DNA solution together with 20 ~1 of water or a freshly prepared solution of ascorbic acid (175 pg/ml). Incubations containing ascorbic acid thus had a S-fold molar excess of ascorbic acid over 6-nitrosochry-

sene.

Parallel incubations were carried out under an argon atmosphere with DNA solutions that had been purged with argon prior to addition of 6-nitrosochrysene and ascorbic acid. Incubations were carried out for 5 h at 37°C. The reaction mixtures were extracted twice with equal volumes of ethyl acetate, twice with equal volumes of n-butanol and brought to a final concentration 0.5 M NaCl before precipitation of DNA with 2 volumes of ethanol. The DNA was washed with 70% ethanol, dried and dissolved in 5 mM bistris, 0.1 mM EDTA, pH 7.1. The concentration of the DNA solution was determined spectrophotometrically and 2 pg portions were analyzed by “P-postlabeling using the n-butanol enrichment procedure described above. An equal amount of DNA from CHO-Kl cells treated with 6-nitrosochrysene was analyzed in parallel with the treated calf-thymus DNA. FoIlowing autoradiography, adduct spots and a background area of the plate were cut, placed in vials with 10 ml Ultima Gold scintillation cocktail (Packard Instrument Co., Downers Grove, IL) and analyzed by scintillation counting. Results

Treatment of CHO-Kl cells with solvent (DMSO, final concentration 1%) alone resulted in 3 + 4 (n = 11) or 4 * 2 (n = i3) mutants per 10’ viable cells in the absence or presence of S9, respectively. Simiiar treatment of CHO-UVS cells yielded 10 + 6 (n = 12) or 11 f 10 (n = 12) mutants per 10” viable ceils in the absence or presence of S9, respectively. Fig. 2 shows the results of the mutagenicity and cytotoxicity assays conducted on CHO-Kl cells and CHO-UVS cells exposed to 6-NC, 6-AC, 6-NC-1,2-dihydrodiol, 6nitrosochrysene, 6-AC-1,Zdihydrodiol and chtysene both in the presence and absence of activation by liver S9 from Aroclor 1254-pretreated rats. The mutagenicity data are summarized in Table 1. In all cases, the mutagenic response was greater in the excision-repair-deficient CHO-UV5 cells. In the absence of S9 activation, only 6nitrosochrysene produced a strong mutagenic re-

157

pgMutagen/ml Fig. 2. Mutagenicity

(top

graphs) and cytotoxicity of &nitrochrysene, h-aminochrysene, 6-nitrosochrysene, 6-nitrochrysene 1.2-dihy1,2-dihydrodiol and chrysene in CHO-KI (0.0) and CHO-WS (A, A 1 cells in the absence (open symbols) and presence (closed symbols) of liver S9 from Aroclor 12.54induced rats.

drodiol, 6-aminochrysene

from CHO-Kl cells treated with 6-NC, 6-AC and their metabolites (Figs. 3-6). n-Butanol extracts of ‘* P-postlabeled nucleotides from [“HI N-hydroxy-6-AC-modified DNA showed 2 major spots (2 and 3, Fig. 3A). In addition, a minor spot was observed in some samples (1, Fig. 3A). These adduct spots were not detected in control DNA (Fig. 3B). The p hosphate buffer used for the D4 chromatography (0.72 M sodium phosphate, 0.45 M Tris, 7.6 M urea, pH 8.2) gave clear separation of the two major adduct spots (spots 2 and 3) whereas the use of 0.75 M lithium chloride, 0.47 M Tris, 8 M urea, pH 8.0 as the D4 buffer did not separate these adducts completely (data not shown). Similar profiles were obtained with DNA from CHO-Kl cells treated with 6-nitrosochrysene in the absence of S9 or with 6-AC in the presence of S9 (Fig. 3C and 3D). Treatment of the MN/SPD digest of the N-hydroxy-6-ACmodified calf-thymus DNA with nuclease Pl prior to labeling with [y- 32P]ATP resulted in one major adduct spot (Fig. 4A) that had a chromatographic mobility corresponding to spot 2 observed with the n-butanol enhancement procedure (Fig. 3A). Identical results were obtained with DNA

sponse at low concentrations, although 6-NC showed weak direct-acting mutagenic activity in CHO-UVS cells. The order of mutagenic potency (mutants per 10” viable cells per nmole of compound per ml, as determined from the increasing portion of the dose-response curve) in the presence of S9 activation was 6-AC-1,2-dihydrodiol > 6-AC > chrysene > 6-NC-1,2-dihydrodiol > 6-NC. The rank order of toxicity of the compounds, as measured by a decrease in the percent relative cloning ability of the cells, generally paralleled the rank order of mutagenic potency. DNA adduct formation in CHO cells treated with bnitrochrysene, baminochrysene, 4-nitrosochrysene, bnitrochrysene-1,2-dihydrodiol and &aminochrysene-1,2-dihydrodiol

“‘P-Postlabeling analyses of calf-thymus DNA modified with N-hydroxy-6-AC or a microsomal metabolite of 6-AC-1,2_dihydrodiol were carried out using either a modification of the n-butanol adduct enrichment procedure (Gupta, 1985) or the nuclease Pl enrichment procedure (Reddy and Randerath, 1986). The resulting profiles were compared with the profiles obtained with DNA

TABLE 1 SUMMARY OF THE MUTAGENIC RESPONSES PRODUCED THEIR METABOLITES IN CHO-Kl AND CHO-UVS CELLS

BY 6-NITROCHRYSENE,

Compound

a

Mutants/IO’

clonable cells/nmole/ml

CHO-Kl

6Nitrochrysene 6Aminochrysene 6Nitrosochrysene 6Nitrochrysene 1.2-dihydrodiol 6-Aminochrysene 1,Zdihydrodiol Chrysene a

Mutagenicityvalues

6-AMINOCHRYSENE

AND

CHO-UV5 .~

-s9

+s9

-s9

+s9

< 0 b to.091 c 0 (0.301 127 (0.99)

0.3 (0.73) 35 (0.94) NDd

0.3 (0.581
4 (0.97) 117 (0.941 ND


(0.17)

1

(0.91)

0.8 <0

(0.82) (0.241

488 12

(0.98) (0.98)

<0

(0.56)

0.6 (0.3 I ) 0 to.201

6 (0.97) 644 (0.96) 28 (0.94)

were calculated from the slopes of the linear regression lines fit to the increasing portions of the curves in Fig. 2. b Dose-responsecurve had negative slope. ’ Numbers in parentheses are correlation coefficients (~‘1 of linear regression lines from which mutagenicity values were calculated. d ND, not determined. dose-response

IS’)

droxy-6-aminochrysene (Delclos et al., 1987a), was added to parallel incubations. The adduct patterns, as determined by ALP-post~abe~ingfollowing the n-butanol enrichment method described in Materials and Methods, were identical to those shown in Fig. 3 for N-hydroxy-6-AC-modified calf-thymus DNA or DNA from 6nitrosochrysene-treated CHO cells. The areas corres~nd~ng to adduct spots 1 and 2 shown in Fig. 3 were cut and quantified as described in Materials and Methods and the results are tabulated in Table 2. Inclusion of ascorbic acid in the incubation mix-

Fig. 3. Autoradiograms of “‘P-postlabeled rr-butanol extracts of enzymatic digests of DNA from CHO cells treated with 6nitrosochrysene (-S91, dimethyl sulfoxide (+S9) or 6aminochrysene ( + S9) and N-hydroxy-6-AC-treated Calfthymus DNA. DNA (2 rgf from the sources indicated below was digested with micrococcal nuclease/spleen phosphodiesterase and extracted with n-butanol as described in Materials and Methods prior to labeling with [Y-~‘P]ATP. (A) Calfthymus DNA modified by reaction with N-hydroxy-6aminochrysene in vitro. (B) DNA isolated from CHO-Kl cells treated with dimethyi sulfoxide in the presence of S9. fC) DNA isolated from CHO-KI cells treated with 1 pg/mt h-nitrosochrysene. (DI DNA isolated from CHO-Kl cells treated with 2.5 pg/ml h-aminochrysene in the presence of S9. The arrows in A indicate the areas of the plate where the three adduct spots were detected. For all of the chromatograms shown in this and subsequent figures, the origin (01 is at the lower lefthand corner of the plate. D3 was run from bottom to top and D4 and DS were run from left to right.

isolated from CHO-Kl cells treated with Ci-nitrosochrysene in the absence of S9 or with 6-AC in the presence of S9 (Fig. 4C and 4D). The question of whether 6nitrosochrysene could bind directly to DNA or required further metabolism to N-hydroxy-6-AC was examined by incubating 6-nitrosoch~sene (1 ~g/ml) with calf-thymus DNA (2 mg/ml) in 20 mM sodium phosphate buffer, pH 7.0. A 5fold molar excess of ascorbic acid, a reducing agent that has been shown to convert 6-nitrosochrysene to N-hy-

Fig. 4. Autoradiograms of “‘P-postlabeled nuclease PI-treated enzymatic digests of DNA from CHO cells treated with hnitrosochrysene (-S9), dimethyl sulfoxide (+S9) or haminochrysene ( + S9) and N-hydroxy-h-AC-treated calfthymus DNA. DNA (I gg) from the sources indicated below was digested with microc~cal nuclease/spleen phosphodi” esterase and treated with nuclease Pl as described in Materials and Methods prior to labeling with [Y-~~P]ATP. (A) Calfthymus DNA modified by reaction with N-hydroxy-haminochrysene in vitro. (B) DNA isolated from CHO-KI cells treated with dimethyl sulfoxide in the presence of S9. (Cf DNA isolated from CHO-Kl cells treated with 1 j&ml 6-nitros~h~sene. (D) DNA isolated from CHO-Kl cells treated with 2.5 &g/ml 6aminochrysene in the presence of S9. DNA was treated with micrococcal nuclease and spleen phosphodiesterase followed by nuclease PI as described in Materials and Methods prior to labeling with [y-“PIATP.

chrysene or N-hydroxy-&AC (greater mobility in D3 and D4 than spots 1 and 2 in Figs 3 and 4). A chromat~graphically identical adduct was identified in DNA isolated from CHO-Kl cells treated with 6AC-1,Zdihydrodiol in the presence of Aroclor-induced rat-liver S9 (Fig. 5E3).

Fig. 3. Autor; iograms of 3’P-postlabeled nuclease R-treated enzymatic digests of DNA from CHO ceils treated with 6-ACI.~-dibydrodioi f+S9) or DNA treated in vitro with &ACl.2-dihydrodiol in the presence of liver microsomes from a 3-methylcholanthrene-pretreated rat. M icrococcal nuclease/spleen phosphodiesterase digests of DNA were treated with nuclease Pl prior to labeling with [v-“P]ATP as described in Materials and Methods. Identical profiles were obtained when the samples were analyzed using the rr-butanol enrichment procedure. (A) Calf-thymus DNA treated in vitro with 6-aminochtysene-1.2dihydrodiol in the presence of liver microsomcs from a 3-methylchoanthrene-pretreated rat. (B) DNA from CHO-KI cells that had been treated with 0.25 *g/ml 6aminochrysene-l.2-dihydrodiol in the presence of liver S9 from Aroclor 1254-pretreated rats.

ture to promote the formation of N-hydroxy-6-AC enhanced the number of adduct counts recovered by approximately 20-fold. Incubation under an argon atmosphere resulted in a 2-fold increase in binding whether or not ascorbic acid was present. As expected from previous studies (Delclos et al., 1988), autoradiograms of 32P-postlabeled DNA from the in vitro incubation of 6-AC-1,2-d& hydr~io~ with calf-thymus DNA in the presence of microsomes from 3-methylcho~anthrene-pretreated rats gave a single treatment-related adduct spot (Fig. 5A) that was chromatographically distinct from the adducts derived from 6-nitroso-

Fig. 6. Autoradiograms of ~‘P-postiabeled ~z-butanol extracts of enzymatic digests of DNA from CHO-Kl celis treated with h-nitrochrysene (+S9) or 6nitrochrysene-1,2-dihydrodiol ( +S9), or from calf-thymus DNA treated in vitro with 6nitrochrysene-1,2-dihydrodiol in the presence of xanthine oxidase and hypoxanthine. Micrococcal nuclease/spleen phosphodiesterase digests of DNA (2 yg) were extracted with n-butanol prior to labeling with [Y-~‘P]ATP as described in Materials and Methods. Identical adduct profiles were obtained when samples were analyzed by the nuclease Pl enrichment procedure. (A) Calf-thymus DNA treated in vitro with 6-nitrochrysene-l,Zdihydrodiol in the presence of xanthine oxidase and hypoxanthine. tBf DNA from CHO-Kl celk treated with S pg/ml 6-nitroch~sene-1,2-dihydrodiol in the presence of S9. (Cl DNA from CHO-Kl cells treated with 15 pg/ml 6-nitrochrysene in the presence of S9. In each panel, the major treatment-related adduct spot referred to in the text is indicated.

lhl TABLE IN

2

VITRO

BINDING

CALF-THYMUS

OF

h-NITROSOCHRYSENE

TO

DNA”

Treatment

Total adduct cpm recovered’

&Nitrosochrysene,

air

233h&

195

h-Nitrosochrysene,

argon

5331+

571

&Nitrosochrysene.

ascorbic acid. air

48 372 f 3 094

6-Nitrosochtysene,

ascorbic acid. argon

97233f2996

” Calf-thymus

DNA

(2 mg/ml

at 37°C for 5 h with or without and 2-pg gested,

portions

n-butanol

matographed h Adduct

(in

duplicate)

extracted,

were

excess of

enzymatically

“P-postlabeled

as described in Materials

and

dichro-

and Methods.

2 (see Fig. 3) were

and radioactivity

scribed in Materials

(I pg/ml)

a 5-fold molar

was isolated from the reaction mixtures

spots 1 and

chromatograms

Discussion

in 20 mM sodium phosphate

buffer, pH 7.0) was treated with 6-nitrosochrysene ascorbic acid. DNA

PAHs to N-hydroxy derivatives (Howard et al.. 1983). Since 6-NC showed a weak direct mutagenic activity in CHO-UV5 cells. DNA was isolated from CHO-UV5 cells that had been exposed to 15 pg/ml 6-NC for 5 h. No clear adduct spots were observed in autoradiograms of “P-postlabeled DNA from these cells (data not shown).

cut from

was quantitated

the

as de-

and Methods. The numbers given are

averages of duplicate determinations+

the range.

‘2P-Postlabeling analyses of DNA from CHOKl cells treated with 6-NC-1,2-dihydrodiol or 6-NC in the presence of S9 indicated a single major treatment-related spot accounting for approximatley 90% of the adduct-associated activity on the plate (Fig. 6, B and 0. In contrast to the adduct pattern found in the DNA of CHO cells treated with 6-AC in the presence of S9 or 6nitrosochrysene without S9. the addduct profile in hydrolysates of DNA from CHO cells treated with 6-NC or 6-NC-1,2-dihydrodiol with S9 was identical when the n-butanol (Fig. 6) or nuclease Pl enrichment methods were used for “‘P-postlabeling. The major adduct spot observed in 6-NCor 6-NC-1,2-dihydrodiol-treated cells (D3 R, = 0.50, D4 R, = 0.55) was chromatographically distinct from the adduct spots seen in the DNA of cells treated with 6nitrosochrysene and 6-AC (Figs. 3 and 4; Spot 2, D3 R, = 0.36, D4 R, = 0.34) or 6-AC-1,2-dihydrodiol (Fig. 5; D3 R, = 0.85, D4 R, = 0.60). An adduct spot with chromatographic mobility identical to the major adduct found in the DNA from cells treated with 6-NC or 6-NC1,Zdihydrodiol was the major product detected detected in calf-thymus DNA modified by incubation with 6-NC-1,Zdihydrodiol in the presence of xanthine oxidase and hypoxanthine (Fig. 6A), an enzymatic system capable of converting nitro

The data presented in this paper demonstrate that 6-AC, 6-nitrososchrysene and 6-AC- 1,Zdihydrodiol are mutagenic in CHO cells. The major mutagen-DNA adducts formed in cells treated with these compounds had chromatographic properties that corresponded to previously reported adducts formed from N-hydroxy-6-AC (Delclos et al., 1987a) or a metabolite of 6-AC1,2-dihydrodiol presumed to be 1,2-dihydroxy3,4-epoxy-1,2,3,4-tetrahydro-6-aminochrysene (Delclos et al., 1988). In addition, the data indicate that under the conditions of the S9-mediated CHO mutagenicity assay, the metabolic activation of 6-NC and 6-NC-1,2-dihydrodiol proceeds by a mechanism that does not involve the formation of N-hydroxy-6-AC or 6-AC- 1,2-dihydrodiol. Cells treated with 6-AC-1,Zdihydrodiol in the presence of S9 contained a single adduct that was presumably derived from the oxidative metabolism of the dihydrodiol to a diol epoxide (Delclos et al., 1988; El-Bayoumy et al., 1989a). The differential response of the repair proficient CHO-Kl cells and the CHO-UV5 cells was less dramatic for 6-AC-1,2_dihydrodiol than for the other test compounds (Table 1). This observation suggests that the adduct derived from 6-AC-1,2dihydrodiol may be more resistant to repair than one or more of the adducts derived from Noxidized derivatives of S-AC. Adducts derived from 6-AC-dihydrodiol have been shown to be formed in mouse lung and liver under the conditions of the tumorigenicity assay of 6-NC (Delclos et al., 1987b, 1988), but the persistence of the adduct or the significance of the persistence has not been evaluated. 6-Nitrosochrysene was highly mutagenic in CHO cells and the pattern of DNA adducts formed in 6-nitrosochrysene-treated cells was

lo’

to that produced from the in vitro iXXCX-hydroky-&AC with calf-thymus DNA (Figs. 3 and 4). The hV0 major and one minor adduct spots observed in these samples presumably contain the three previously described adducts formed from the reaction of N-hydroxy6-AC with calf-thymus DNA (Delclos et al., 19S7a). The direct-acting mutagenicities of several nitroso derivatives of nitro PAHs (I-nitrosopyrene. I-nitro-6-nitrosopyrene, 1-nitro-8nitrosopyrene, I-, 3- and &nitorosobenzo[aIpyrene) in CHO cells have been previously demonstrated and attributed to the ability of the cells to reduce each of the nitroso derivatives to the corresponding A!-hydroxy derivative (Fifer et al., 1986: Heflich et al., 1985a, 1986a,b, 1989). The conclusion that nitrosoarenes require conversion to IV-hydroxyarylamines to exert mutagenic responses is based on several observations. First, nitrosoarenes have low reactivity with DNA relative to N-hydroxyarylamines or their esters (Kadlubar and Beland, 1985; Heflich et al., 1985bI. Second, the adducts found in the DNA of ceils treated with 1-nitrosopyrene are identical to those expected from N-hydroxy-1-aminopyrene (Heflich et al., 1985b, 1986a). Third, incubation of 1-nitrosopyrene with CHO cells results in the nearly compIete conversion of 1-nitrosopyrene to I-aminopyrene, which indicates the capacity of CHO cells to carry out reductive meta~iism of the nitroso derivative through a pathway that involves the intermediate formation of N-hydroxy-1-aminopyrene (Heflich et al., 1986a). We have sho& that, like I-nitrosopyrene (Heflich et ai., 1985b), 6-nitrosochrysene reacts with calfthymus DNA without the addition of a reducing agent, but that the extent of reaction is approximately 20-fold lower than that observed when the incubation is carried out in the presence of ascorbic acid, a reducing agent that has been shown to convert 6-nitrosoch~sene to ~-hydroxy-6aminochrysene (Delclos et al., 1987a). Although we did not examine the reductive metabolism of 6-nitrosochrysene in the CHO incubations, it is likely that the compound is metabolized similarly to 1-nitrosopyrene and that conversion to IV-hydroxy-6-AC and 6-AC occurs. 6-NC showed a weak direct-acting mutagenic activity in CHO-UVS cells. It had been suggested i&ntic;tl

tion

of

previously that l-nitropyrene lacked mutagenicity in CHO cells because of the absence or limited activity of a nitroreductase capable of metabolizing the compound to N-hydroxy-1-aminopyrene or because the Iesion formed was not mutagenic in these cells (Eddy et al., 1986; Heflich et al., 1986a). However, a more recent study indicates that extended exposure (24 h vs. 5 h) of repairdeficient CHO-UV5 cells to 1-nitropyrene results in a significant mutagenic response and mutagenDNA adducts derived from N-hydroxy-laminopyrene (Thornton-Manning et al., 1991). In the present case, a weak mutagenic response was detected in CHO-UV5 cells, but not in CHO-Kl cells, after a 5-h exposure to 6-NC. Although the mutagenic response observed in the repair-deficient cells implies that DNA damage was induced by treatment with 6-NC, we were not able to confirm the presence of adducts derived from metabofites of 6-NC and were thus unable to establish the pathways responsible for the observed direct-acting mutagenicity. Both &NC and 6-NC-1,2-dihydrodiol produced weak, S9-mediated mutagenic responses that were greatly enhanced in the excisionrepair-deficient CHO-UV5 cells. The predominant adducts formed in these cells were chromatographically identical and distinct from adducts formed from pathways involving the intermediate formation of N-oxidized derivatives of 6-AC (6-nitrosochrysene and, possibly, N-hydroxy-6-AC) or &AC- 1,Zdihydrodiol. The detection of a chromatographicaliy similar adduct in caIf-thymus DNA treated in vitro with 6-NC-1,2dihydrodiol in the presence of xanthine oxidase and hypoxanthine, a mammalian nitroreduction system, supports the formation of ~-hydro~-6AC-1,2-dihydrodioi as the activation pathway for both 6-NC and 6-NC-1,2-dihydrodiol in CHO cells in the presence of S9. In contrast to the results reported here, DNA from Salmonella treated with 6-NC in the presence of Aroclor-induced S9 contained, as the predominant adduct, an adduct that was chromatographically indistinguishable by HPLC analysis from the adduct produced by the presumed 1,2-dihydroxy3,4-epoxy-1,2,3,4-tetrahydrod-AC (El-Bayoumy et al., 1989b). The present results confirm the biological activity of 6-AC-1,2-dihydrodiol and N-oxidized

derivatives of &AC and demonstrate further complexity to the metabolic activation pathways available to &NC and &AC. The nature of the mutations induced by &NC and its derivatives and the roles of individual adducts in producing mutations and other toxicological effects of these compounds remain to be determined.

The expert technical assistance of Ralph P. Walker, Toni Y. Minor and Tammie A. McRae is gratefully acknowledged. Initial experiments on the “P-postlabeling of carcinogen-DNA adducts derived from metabolites of 6-NC were carried out by Dr. Glenn Talaska. We also thank Letha H. Couch for help in setting up the ““P-postlabeling procedure. References Beland, F-A.. and F.F. Kadlubar (1990) Metabolic

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