identification and mutagenicity of metabolites of 1 ...study indicate that both nitroreduction and...

[CANCER RESEARCH 43, 3132-3137, July 1983]

Identification and Mutagenicity of Metabolites of 1-Nitropyrene Formedby Rat Liver1

Karam EI-Bayoumy2 and Stephen S. Hecht

Division of Chemical Carcinogenesis, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York, 70595

ABSTRACT

The metabolism of 1-nitropyrene by rat liver 9000 x g super

natant was investigated. Under aerobic conditions, ring oxidationto 1-nitropyren-3-ol, 1-nitropyren-6-ol, 1-nitropyren-8-ol, and 4,5-dihydro-4,5-dihydroxy-1 -nitropyrene and nitroreduction to 1-ami-

nopyrene were observed. Metabolites were identified by theirultraviolet, mass, and nuclear magnetic resonance spectra; bychemical transformations; and by comparison to reference standards. When incubations were carried out in an atmosphere of4% O2 in N2, 1-aminopyrene was the major metabolite. Themutagenic activities of 1-nitropyren-3-ol, 1-nitropyren-6-ol, and1-nitrosopyrene were assessed in Salmonella typhimurium

strains TA 98 and TA 100. In strain TA 98, without activation,doses of 0.5 /Â¿g/plateor less of these three compounds weremore mutagenic than was 1-nitropyrene; however, their activities

decreased rapidly at higher doses. In the presence of rat liver9000 x g supernatant, they were less mutagenic than was 1-

nitropyrene at all doses tested. In S. typhimurium TA 100, withoutactivation, 1-nitropyren-3-ol, 1-nitropyren-6-ol, and 1-nitrosopyrene were more mutagenic than was 1-nitropyrene at doses of

0.25 ^g/plate or less, but their activities decreased at higherdoses. In strain TA 100, with activation, only 1-nitropyren-6-olwas more mutagenic than was 1-nitropyrene. The results of this

study indicate that both nitroreduction and ring oxidation maybe involved in the mutagenic activity of 1-nitropyrene.

INTRODUCTION

1-Nitropyrene is a potentially important environmental pollutant

because of its high mutagenicity toward Salmonella typhimuriumand its presence in diesel exhaust (15, 20, 22). The projectedrise in the use of diesel-powered automobiles has encouragedmore thorough studies on the biological properties of 1-nitropyrene. Two assays of 1-nitropyrene for tumorigenicity have been

reported. It induced s.c. sarcomas upon injection in male F344/DuCrj rats but was inactive as a tumor initiator on the skin offemale CD-1 mice (6, 19). Whereas the exceptional direct-actingmutagenicity of 1-nitropyrene toward S. typhimurium appears to

be due primarily to nitroreduction and possibly to the formationof specific DMA adducts derived from an intermediate hydroxyl-

amine or nitrenium ion (12, 16), little is known about the mammalian metabolism of 1-nitropyrene. As in the case of unsubsti-

tuted and methylated polynuclear aromatic hydrocarbons, specific metabolites such as epoxides, dihydrodiols, or phenols arelikely to be important in determining its potential carcinogenicity,

but the structures of these key products of ring oxidation hadnot been previously determined. In the present study, we haveidentified the major metabolites of 1-nitropyrene formed uponincubation with rat liver supernatant under aerobic and oxygen-

deficient conditions and have assessed the mutagenic activitiesof selected metabolites toward S. typhimurium.

MATERIALS AND METHODS

Apparatus. Melting points were determined with a Thomas-Hoovercapillary melting point apparatus and are uncorrected. HPLC3 was per

formed with a Waters Associates Model ALC/GPC-204 high-speed liquid

Chromatograph equipped with a Model 6000A solvent delivery system,a Model U6K septumless injector, and a Model 440 UV/visible detector.Three reverse-phase columns were used in this work: a 9.4-mm x 50-cm Magnum 9, Partisil-10 ODS-2 column, Whatman Laboratory Products, Inc. Clifton, N. J. (Column 1); a 6-mm x 30-cm da-^Bondapakcolumn, Waters Associates, Milford, Mass. (Column 2); and a 4.6-mm x12.5-cm HS-5|um C,e column, Perkin-Elmer Corp., Norwalk, Conn. (Col

umn 3). TLC was performed on Silica Gel 60 F254glass plates (EMLaboratories, Inc., Elmsford, N. Y.). For the analysis of head-space gases(O2 and N2), we used a Hewlett-Packard Model 7620A gas Chromatograph equipped with a thermal conductivity detector and a 5-ft x 0.125-

inch MS 5A column. The conditions were: column temperature, ambient;injector temperature, 175Â°; detector temperature, 220Â°; and flow rate

(helium), 30 ml/mm. Capillary GC was carried out with a Hewlett-Packard

Model 5830A instrument equipped with a Model 18835B capillary inletsystem and a 20-ft Dexsil 300 glass column, temperature programmedfrom 100-240Â° at 2Â°/min. High-resolution MS analysis was performed

by Shrader Analytical and Consulting Laboratories, Detroit, Mich. UVspectra were determined with a Cary Model 118 instrument. PMRspectra were recorded on a Hitachi Perkin-Elmer R-24 high-resolution

spectrometer and on a Nicolet Magnetics NTC 300 Wide Bore spectrometer at Rockefeller University, N. Y.4 The proton resonance shifts were

measured relative to tetramethylsilane and are expressed in ppm on theÃ³scale.

Chemicals. Glucose 6-phosphate, NADP+, and potassium phosphate

buffer (pH 7.4) were obtained from Sigma Chemical Co. (St. Louis, Mo).1-Aminopyrene, 1-nitropyrene, and phenanthrene were obtained from

Aldrich Chemical Co., Inc. (Milwaukee, Wis.).1-Nitropyrene. The commercial sample was chromatographed on

silica (Silicar CC-7) with elution by 10% ethyl acetate:hexane. The purity

of the compound was >99% based on TLC (silica, hexane:ether, 3:1),HPLC analysis (Column 2, a linear gradient from 45% methanol:H2O to80% methanol:H2O in 1 hr at 2 ml/min; Column 3, a linear gradient from60% methanol:H2O to 80% methanol:H2O in 30 min at 1 ml/min), andcapillary GC. MS m/e was 247 (M+, 100), 217 (51), 201 (87), 200 (68),

and 189 (50). PMR (300 MHz) and the above-mentioned Chromatographie

analyses indicated the absence of dinitropyrenes.Mononitrophenanthrenes. 1-Nitrophenanthrene and 3-nitrophenan-

' This study was supported by National Institute of Environmental Health Sciences Award ES-02477. Presented in part at the 75th Annual Meeting of theAmerican Association for Cancer Research, St. Louis, Mo., April, 1982 (7). This isPaper 52 of the series, "A Study of Chemical Caranogenesis."

2To whom requests for reprints should be addressed.

Received November 18,1982; accepted April 5,1983.

3The abbreviations used are: HPLC, high-pressure liquid chromatography; TLC,thin-layer chromatography; MS, mass spectrum; GC, gas chromatography; PMR,proton magnetic resonance; THF, tetrahydrofuran; DMSO, dimethyl sulfoxide.

* The NTC Wide Bore spectrometer at the Rockefeller University was purchasedwith funds from the National Science Foundation (PCM-7912083), the Camille andHenry Dreyfus Foundation, and the Fleischmann Foundation.

3132 CANCER RESEARCH VOL. 43

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Metabolites of 1-Nitropyrene

threne were obtained by nitration of phenanthrene as described previously (3). 1-Nitrophenanthrene had a melting point of 135Â°, [literature(3), 133Â°], and 3-nitrophenanthrene had a melting point of 174-176Â°[literature (3), 172-174Â°]. The purity of each Â¡somerwas established by

HPLC (Column 2; isocratic, 65% methanol:H2O, 2 ml/min); 1-nitrophen-anthrene eluted at 13 min and 3-nitrophenanthrene eluted at 16 min.

1-Nitropyrenols. 1-Acetoxypyrene was obtained by treatment of py-

rene with lead tetraacetate in refluxing benzene:acetic acid (9:1) for 6 hr.The crude material was chromatographed on silica gel with elution by10% benzene:hexane and recrystallized from 10% ethyl acetate:hexane,m.p. 104-105Â° [literature (24), 102Â°]. Nitration of 1-acetoxypyrene wasperformed as described previously (1) using HNO3 in CH3COOH at 30Â°.

The crude mixture of the acetoxynitropyrenes was treated with CH3ONain 50% methanol-.THF for 20 min at room temperature to afford a mixture

of nitropyrenols. This mixture was chromatographed on preparative TLC(silica, 20 cm x 20 cm x 2 mm; 20% ethanol:benzene) to give a majorband (R( 0.48) and a minor band (R( 0.58). The major band was a mixtureof 70% 1-nitropyren-6-ol and 30% 1-nitropyren-8-ol. A pure sample of 1-nitropyren-6-ol was obtained by collection from HPLC (Column 1, 65%methanol:H2O for 20 min followed by a 1-hr linear gradient to 95%methanol:H2O at 3 ml/min. Retention volumes: 1-nitropyren-8-ol, 197.4ml; and 1-nitropyren-6-ol, 201 ml). The minor band was a single Â¡somer,1-nitropyren-3-ol (m.p. 258Â° with decomposition). A small amount of a

dinitropyrenol was also detected but was not investigated further.Spectral data for 1-nitropyren-6-ol and 1-nitropyren-3-ol were as fol

lows. For 1-nitropyren-6-ol, MS (m/e) was 263 ((vT, 100), 233 (39), 217(53), and 187 (34), and PMR (DMSO-cfe) was 11.30 (bs, OH), 8.74 to8.06 (m, 7H), and 7.72 (d, J = 8.30 Hz, H7); for 1-nitropyren-3-ol, MS (mle) was 263 (M+, 100), 233 (26), 217 (66), and 187 (35), and PMR (DMSO-

d6) was 11.47 (bs, OH), 8.61 (d, J = 9.44 Hz, H10),8.46 to 8.24 (m, 5H),8.23 (s, H2), and 8.15 (t, J = 7.63 Hz, H7). HPLC retention volumes were

as follows. For Column 2, with a linear gradient from 45% methanol:H2Oto 80% methanol:H2O in 1 hr at 2 ml/min: 1-nitropyren-6-ol, 72 ml; and1-nitropyren-3-ol, 81 ml; for Column 3, with a linear gradient from 60%methanol:H2O to 80% methanol:H2O in 30 min at 1 ml/min: 1-nitropyren-6-ol, 19 ml; and 1-nitropyren-3-ol, 21 ml. Additional structural informationwas provided by chemical transformations. 1-Nitropyren-6-ol (5 mg) was

dissolved in 10 ml of 50% methanol:THF, and 1 ml of 20% NH4CI solutionwas added. Ten mg of zinc dust were added with vigorous stirring atroom temperature. After 20 min, the reaction mixture was filtered, and30 ml of H2O were added. The product was extracted with 3 x 20 ml ofethyl acetate, and the extracts were dried (MgSO4) and concentrated.The aminopyrenol had a characteristic fluorescence and a R( of 0.34(20% ethanol in benzene). Without further purification, 1 ml of methanolwas added to the aminopyrenol, followed by 1 ml of concentrated HCI.To this solution, a freshly prepared solution of 0.8 g of FeCI3 in 0.5 ml ofconcentrated HCI and 1 ml of H2O was added with vigorous shaking (9).As the reaction progressed, the characteristic fluorescence of the aminopyrenol disappeared. After 10 min, the reaction mixture was dilutedwith 10 ml of H2O and extracted twice with 20 ml of ethyl acetate. Thesolvent was dried (MgSO4) and evaporated to give 1,6-pyrenequinone,

which had a R, of 0.60 (20% ethanol in benzene) and UV identical tothose of a standard. The same sequence was carried out on a mixtureof 1-nitropyren-6-ol and 1-nitropyren-8-ol to give a mixture of 1,6-pyrenequinone and 1,8-pyrenequinone (R( of 0.54; 20% ethanol in benzene).For 1-nitropyren-3-ol, the same sequence gave an aminopyrenol (Rf 0.39;

20% ethanol in benzene) which did not undergo oxidation.Pyrenequinones. Pyrene (50 mg, 0.24 mmol) was allowed to react

with CrO3 (72 mg, 0.72 mmol) in 80% aqueous CH3COOH at 0Â°for 3 hr

and then at room temperature for 72 hr as described previously (4). Thereaction mixture was poured into 200 ml of H2O and then extracted withethyl acetate (3 x 25 ml). The organic layer was dried (MgSO4) andconcentrated to give a mixture of pyrenequinones (10 mg, 0.04 mmol,17%) which was separated by preparative TLC on 20-cm x 20-cm 0.5-

mm silica plates with elution by 20% ethanol in benzene. This yielded1,6-pyrenequinone, R( 0.60, 65% of the mixture, yellow needles, meltingpoint 309-311Â° [literature (24), 309Â°], MS m/e 232 (M+, 100), 204 (26),

and 176 (68) and 1,8-pyrenequinone, R( 0.54, 35% of the mixture, orangeneedles, melting point 271-273Â° [literature (24), 270Â°,MS m/e 232 (M*,

100) 204 (25), and 176 (59). The UV spectra of both isomers wereidentical to those reported previously (18).

1-Nitrosopyrene. A solution of 1-aminopyrene (108 mg, 0.49 mmol)in 100 ml of CH2CI2 was cooled to 0Â°,and m-chloroperbenzoic acid (173

mg, 1 mmol) in 25 ml of CH2CI2 was added dropwise under a N2atmosphere. After addition of the peracid, stirring was continued for 20min. The reaction mixture was washed with 1 N NaOH, H2O, 6 N HCI,arid H2O, dried (MgSO4), and concentrated. The crude product waspurified by preparative TLC in the dark (silica, 20 cm x 20 cm x 2 mm;hexane:ether, 3:1) to give 1-nitrosopyrene (35 mg, 0.15 mmol, 31%),melting point 146-148Â° (benzene:hexane), MS m/e, 231 (M+, 42), 202

(20), 201 (100), and 200 (48), and PMR (CDCI3), a 9.75 (d, J = 9 Hz, H2),8.15 to 7.40 (m, 7H), and 6.50 (d, J = 10 Hz, H)0). High resolutionMS:C16H9NO.

C,6H9NO

Calculated: 231.0683Found: 231.0692

Metabolism in Vitro. Incubations were carried out in 50-ml Erienmeyerflasks containing 20 ml of S-9 mix and 1.0 mg of 1-nitropyrene whichhad been dissolved in 200 n\ of DMSO. The ratio of S-9 mix:substratewas based on mutagenicity data; i.e., 10 ^g of 1-nitropyrene and S-9

mix (200 ^I/plate) gave over 1000 revenants in both strain TA 100 andstrain TA 98. The S-9 mix contained, per ml, 100 Â¿imolof MgCI2, 1.65^mol of KCI, 5 (/mol of glucose 6-phosphate, 4 //mol of NADP+, and 0.5

ml of 9000 x g supernatant (33 mg of protein/ml) from livers of male F-344 rats (250 to 300 g) which had been given i.p. injections of Aroclor1254 (500 mg/kg) in corn oil 5 days prior to sacrifice. The preparation ofthe 9000 x g supernatant has been described previously (8). Reactionswere quenched after 1 hr by addition of 20 ml of ice-cold absolute

ethanol. The protein was filtered, and the ethanol solution was dilutedwith 50 ml of distilled H2O and extracted with 3 x 50 ml of ethyl acetate.The extracts were dried (MgSO4) and evaporated under reduced pressure at 35Â°.The residue was dissolved in 500 ^l of THF, and 50 ;il were

analyzed by HPLC using Column 3 and a linear gradient from 60%metnanol:H2O to 80% methanol:H2O in 30 min with a flow of 1 ml/min.For separation of 1-nitropyren-6-ol and 1-nitropyren-8-ol, the peak eluting

at 19 min was collected and analyzed on Column 1 using the sameprogram used for the synthetic compounds.

For isolation of 4,5-dihydro-4,5-dihydroxy-1-nitropyrene, the incubations were carried out on a preparative scale with 18 mg of 1-nitropyreneand 360 ml of S-9 mix in two 500-ml Erienmeyer flasks for 1 hr at 37Â°.

After workup, the diol (=250 nQ) was collected from HPLC using Column

1, linear gradient from 65% methanol:H2O to 100% methanol:H2O in 30min with a flow of 5 ml/min.

Control incubations were performed as described above, except thatheat-denatured S-9 mix was used. For incubations in a N2 atmosphere,S-9 mix was added to a Reactiflask (Pierce Chemical Co., Rockford, III.)

and flushed with N2 prior to addition of substrate. The amounts of O2and N2 in the head space were determined by GC analyses.

Mutagenicity Assays. S. typhimurium strains TA 98 and TA 100 werekindly provided by Dr. Bruce Ames of the University of California,Berkeley. The procedure of Ames ef al. (2) was used in performing theseassays as described previously (8). The purities of 1-nitropyrene and the

metabolites tested were more than 99% based on HPLC and TLCanalyses.

RESULTS

1-Nitropyrene was incubated aerobically with the 9000 x gsupernatant from the livers of Aroclor-pretreated rats. The ethylacetate-soluble metabolites, which were not observed in control

incubations, were separated by HPLC as illustrated in Chart 1.The indicated peaks were collected and identified by their spec-

JULY 1983 3133



K. EI-Bayoumy and S. S. Hecht

trai properties or by comparison to standards. The peak elutingat 10.3 min had a molecular ion of m/e 281 with a major ion atm/e 263 (M+-H2O). Fragment ions were also observed at m/e

247, 233, 217, 189, and 176. Its UV spectrum was similar tothat of 1-nitrophenanthreneand somewhat different from that of3-nitrophenanthrene, as illustrated in Chart 2. These data indicated that the metabolite was a K-region dihydrodiol of 1-nitropyrene. Its 300-MHz PMR spectrum allowed us to assignthe structure as frans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene.The PMR spectrum showed 2 coupled doublets (J = 7 Hz) at 54.25 and d 4.27, which were assigned as H4and H5in the trans-

0 5 IO IS 20 25MIN

Chart 1. Chromatogram obtained by HPLC analysis on Column 3 of metabolitesformed upon aerobic incubation of 1-nitropyrene with the 9000 x g supernatantfrom livers of rats treated with Aroclor 1254. The peak eluting at 19 min wascomprised of 80% 1-nitropyren-6-ol and 20% 1-nitropyren-8-ol, as determined byanalysis on Column 1.

200 240 360 400280 320WAVELENGTH(nm)

Chart 2. UV spectra of 4,5-dihydro-4,5-dihydroxy-1-nitropyrene (â€¢phenanthrene ( ), and 3-nitrophenanthrene ( ).

configuration, based on the coupling constant. Doublets at Ã³8.15 and 6 8.26 (J = 9 Hz) were assigned to H9and H10,and adoublet at Ã³8.37 (J = 8 Hz) was assigned to H2.These assignments are consistent with the expected deshielding effect of thenitro group. The remaining resonances were assigned, with theaid of decoupling, as follows: a multiplet at 5 7.97 (H3and H7);and doublets at O7.79 and 6 7.90, J = 7 Hz (H6 or H8).Theobserved resonances could possibly have been consistent, however, with the 9,10-dihydrodiol of 1-nitropyrene. If this were thecase, then the 4- and 5-protons should have resonated in positions similar to the 9- and 10-protons of 3-nitrophenanthrene. A300-MHz PMR spectrum of the latter showed that the 9- and10-protons appeared at b 7.7 to 7.8. The only protons withchemical shifts greater than 8 ppm in the spectrum of 3-nitrophenanthrene were H2,adjacent to the nitro group, and H4andH5, the bay-region protons. Since the latter are not present inthe corresponding pyrene derivative, but 3 resonances at chemical shins greater than 8 ppm were still observed, the data arenot consistent with the 9,10-dihydrodiol of 1-nitropyrene. Absolute confirmation of the structure of frans-4,5-dihydro-4,5-dihy-droxy-1-nitropyrene, however, can be achieved only by chemicalsynthesis.

The peak eluting at 14.7 min was identified as 1-aminopyreneby comparison of its HPLC retention time, MS, and UVspectrumto those of a reference standard. A major peak formed in theaerobic metabolism of 1-nitropyrene eluted at 19 min (Chart 1).Upon reanalysis of this peak on Column 1, it was found to be amixture of two 1-nitropyrenols in the ratio of 4:1. The majorcomponent of this peak had HPLC retention time on 3 differentcolumns and a MS and UV spectrum identical to those of themajor product obtained upon nitration of 1-acetoxypyrene followed by hydrolysis (Chart 3). The structure of this compound

220 260 300 340

WAVELENGTH(nm)

380

â€¢),1-nttro- ChartS. UV spectra of metabolic and synthetic 1-nitropyren-6-ol under neutralâ€”) and basic ( ) conditions.





was confirmed as 1-nitropyren-6-ol by reduction with zinc andNH4CI to 1-aminopyren-6-ol, followed by oxidation with FeCI3and HCI to 1,6-pyrenequinone. In a similar way, the minor component of the peak eluting at 19 min was identified as 1-nitropyren-8-ol.

The peak eluting at 21 min had a molecular ion of m/e 263 inits MS, indicating that it was also a 1-nitropyrenol. Its retention

time in 2 different HPLC systems and its UV spectrum wereidentical to those of one of the products formed upon nitrationof 1-acetoxypyrene, followed by hydrolysis (Chart 4). The 300-

MHz PMR spectrum of that product had a triplet at 6 8.15, whichis consistent with 1-nitropyren-3-ol. Only 3 identical nitropyrenolisomers can be obtained from both 1-nitropyrene and 1-acetoxypyrene; these are 1-nitropyren-3-ol, 1-nitropyren-6-ol, and 1-nitropyren-8-ol. 1-Nitropyren-6-ol and 1-nitropyren-8-ol werecharacterized as described above and in "Materials and Methods." Therefore, the peak eluting at 21 min could only be 1-

nitropyren-3-ol.In contrast to the results described above, incubation of 1-

nitropyrene with rat liver 9000 x g supernatant but in an atmosphere of 4% O2 in N2 gave 1-aminopyrene as the major metab

olite (Chart 5).The mutagenic activities toward S. typhimurium TA 98 and TA

100 of 1-nitropyrene, 1-nitropyren-3-ol, 1-nitropyren-6-ol, and 1-

nitrosopyrene, a likely intermediate in the metabolic reduction of1-nitropyrene, are illustrated in Charts 6 to 9. We did not testeither 1-nitropyren-8-ol or 4,5-dihydro-4,5-dihydroxy-1-nitropyrene, which were not available in sufficient quantities, or 1-aminopyrene, which had been assayed previously (14). In strainTA 98, without activation, doses of 0.5 Â¿Â¿g/plateor less of 1-nitrosopyrene, 1-nitropyren-3-ol, or 1-nitropyren-6-ol were more

220 260 300 340

WAVELENGTH(nm)

380

0 10 20 30 40 50MIN

Chart 5. Chromatogram obtained by HPLC analysis on Column 2 of metabolitesformed upon incubation of 1-nitropyrene with the 9000 x g supernatant from liversof rats treated with Arodor 1254. Incubations were carried out in an atmosphereof 4% O2 in N2.

2200

1400

1000

600.

200

0.5 1.0 1.5DOSE(/ug/PLATE)

2.0 25

Chart 4. UV spectra of metabolic and synthetic 1-nitropyren-3-ol under neutralâ€”) and basic ( ) conditions.

Charte. Mutagenicity of 1-nitropyrene (â€¢),1-nitropyren-3-ol (A), 1-nitropyren-6-ol (D), and 1-nitrosopyrene (O) toward Salmonella typhimurium TA 98, withoutactivation. Each point is the mean of 3 determinations. Background revenants (30/plate) have been subtracted.

mutagenic than were the corresponding levels of 1-nitropyrene.

At doses of 1.0 Â¿/g/plateor greater, the mutagenic activities ofthe 3 metabolites were less than that of 1-nitropyrene. None of

the compounds was toxic to the bacteria in the range of dosesexamined. In the presence of rat liver 9000 x g supernatant, the3 metabolites were all less mutagenic toward S. typhimurium TA98 than was 1-nitropyrene. In strain TA 100, without activation,

all 4 compounds were less mutagenic than in strain TA 98.However, as observed in strain TA 98, the metabolites weremore mutagenic than was 1-nitropyrene at the lower doses butless mutagenic than was 1-nitropyrene at the higher doses. As

in strain TA 98, no toxicity was observed in the dose rangestudied. In the presence of rat liver 9000 x g supernatant,significant mutagenicity toward strain TA 100 was observed onlyat the highest dose of 1-nitropyren-6-ol.

DISCUSSION

The major objective of the present study was to identifymammalian metabolites of 1-nitropyrene and to assess their role

JULY 1983 3135



K. EI-Bayoumy and S. S. Hecht

400

Â£300

200

100

10 1.5DOSE(jug/PLATE)

25

Chart?. Mutagenicity of 1-nitropyrene (â€¢),1-nitropyren-3-ol (A), 1-nitropyren-6-ol (D), and 1-nitrosopyrene (O) toward S. typhimurium TA 98, in the presence of9000 x g supernatant from the livers of rats pretreated with Aroclor 1254. Eachpoint is the mean of 3 determinations. Background revertants (36/plate) have beensubtracted.

1.0 1.5DOSE(M9/PLATE)

Charts. Mutagenicity of 1-nitropyrene (â€¢),1-nitropyren-3-ol (A), 1-nitropyren-6-ol P), and 1-nitrosopyrene (O) toward S. typhimurium TA 100, without activation.Each point is the mean of 3 determinations. Background revertants (138/plate)have been subtracted.

1.0 1.5

DOSE(jug/PLATE)

2.0 2.5

Chart 9. Mutagenicity of 1-nitropyrene (â€¢),nitropyren-3-ol (A), 1-nitropyren-6-ol(O), and 1-nitrosopyrene (O) toward S. typhimurium TA 100, in the presence of9000 x g supernatant from the livers of rats pretreated with Aroclor 1254. Eachpoint is the mean of 3 determinations Background revertants (134/plate) havebeen subtracted.

in its mutagenicity. Therefore, we used the 9000 x g supernatantfrom livers of Aroclor-pretreated rats, since this preparation givesmetabolites in sufficient quantities for spectral characterizationand is routinely used in mutagenicity assays. Under aerobicconditions, the major metabolites of 1-nitropyrene were formedby ring oxidation, although some 1-aminopyrene was detected.When the level of oxygen, an inhibitor of nitroreductase activity,was reduced, 1-aminopyrene was the major metabolite. Thus,

the levels of nitroreduction and ring oxidation of 1-nitropyrene invivo could be influenced by oxygen concentration. The relativeextents of metabolic ring oxidation and nitroreduction of nitro-polynuclear aromatic hydrocarbons are also likely to depend onstructure. Underconditions similar to ours, ring oxidation but notnitroreduction was observed in the metabolism of 6-nitro-benzo(a)pyrene(10). In parallel studies on the metabolism of 6-nitrochrysene, we have found that it is less readily reduced thanis 1-nitropyrene (5).

The formation of 1-nitropyren-3-ol, 1-nitropyren-6-ol, 1-nitro-pyren-8-ol, and 4,5-dihydro-4,5-dihydroxy-1-nitropyrene as metabolites of 1-nitropyrene is analogous to the results of earlierstudies on the metabolism of pyrene, in which major metaboliteswere identified as 1-hydroxypyrene and 4,5-dihydro-4,5-dihy-droxypyrene (13, 23). Since 1-nitropyrene is unsymmetrical, di-hydrodiol formation at the 9,10-positions might also have beenexpected. However, this process may have been inhibited by thepresence of the nitro group at the adjacent peri position. Theapparent inhibition of dihydrodiol formation by a peri-nitro grouphas also been observed in studies on 6-nitrobenzo(a)pyreneand6-nitrochrysene (5, 10).

The results of the mutagenicity assays in S. typhimurium TA98 and TA 100, without activation, clearly demonstrate that 1-nitrosopyrene, 1-nitropyren-3-ol, and 1-nitropyren-6-ol have highintrinsic mutagenic activities. The dose dependency of the mutagenicity of these compounds requires further study. The interpretation of this phenomenon is complicated by the presence inS. typhimurium of a family of nitroreductase enzymes with different substrate specificities (21). In addition, reduction of the nitrogroup of the 1-nitropyrenols to the corresponding hydroxyl-amines, which presumably are responsible for the observedmutations, occurs in at least 2 stages. At present, nothing isknown about the kinetics of reduction of these nitro compoundsto hydroxylamines or about their further reduction to the corresponding amines, which would likely be nonmutagenic in theabsence of an activating system. However, the intrinsic mutagenic activities of 1-nitrosopyrene, 1-nitropyren-3-ol, and 1-nitro-pyren-6-ol are in line with previous studies which have shownthat S. typhimurium can activate 1-nitropyrene by reduction (15,16,20-22).

While 1-nitropyren-3-ol, 1-nitropyren-6-ol, and 1-nitrosopyrenewere potent mutagens in the absence of rat liver 9000 x gsupernatant, they were generally less mutagenic than was 1-nitropyrene in the assays carried out with activation. The exception was 1-nitropyren-6-ol, which was more mutagenic than was1-nitropyrene at the highest dose tested in strain TA 100. Therelatively low mutagenic activities of these compounds in thepresence of rat liver 9000 x g supernatant could result partiallyfrom reaction of the intermediate hydroxylamines or nitrosocompounds with nucleophilic components of the activating system. Further studies are necessary to explore this phenomenon.The available data, therefore, do not allow us to unambiguouslyassess the role of the 1-nitropyrenols in the mutagenicity of 1-nitropyrene with activation. However, it is clear that they cannotbe regarded exclusively as detoxification products, as is usuallythe case with unsubstituted polynuclear aromatic hydrocarbons.In the case of 6-nitrobenzo(a)pyrene, the 1-hydroxy and 3-hydroxy derivatives appear to be major proximate mutagens(10).

The high mutagenicity of 1-nitrosopyrene suggests that, ifformed at the site of injection, it may be involved in the induction





of s.c. tumors by 1-nitropyrene (19). Several aromatic C-nitrosocompounds, including 2-nitrosofluorene, o-nitrosotoluene, and3,2'-dimethyl-4-nitrosobiphenyl, are known to induce s.c.tumors5 (11, 17). However, ring oxidation is clearly a majorprocess in hepatic 1-nitropyrene metabolism, and products derived from 1-nitropyren-6-ol and 4,5-dihydro-4,5-dihydroxy-1-nitropyrene may be dominant in vivo. It will be important todetermine their roles in the tumorigenicity of 1-nitropyrene.

ACKNOWLEDGMENTS

The authors thank Dr. D. Hoffmann for his support and advice and Dr. TomikoShimada and Albertina Swanson for carrying out the mutagenicity assays. Thetechnical assistance of M. O'Donnell and R. Morse is appreciated. We also thank

J. Camanzo for providing MS data. We deeply appreciate the continuous help ofour secretarÃa!staff.

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JULY 1983 3137



1983;43:3132-3137. Cancer Res Karam El-Bayoumy and Stephen S. Hecht Formed by Rat LiverIdentification and Mutagenicity of Metabolites of 1-Nitropyrene

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