metabolism and dna binding of 2-nitropyrene in the rat1 · anosin-8-yl)-l-aminopyrene; hplc,...

[CANCER RESEARCH 52, 1176-1181, March I, 1992]

Metabolism and DNA Binding of 2-Nitropyrene in the Rat1

Pramod Upadhyaya, Ajit K. Roy, Peter P. Fu,2 and Karam El-Bayoumy3

American Health Foundation, Valhalla, New York 10595

ABSTRACT

2-Nitropyrene (2-NP), a contaminant of ambient air, is a potentbacterial mutagen in the Ames assay and induces leukemia/lymphoma infemale Sprague-Dawley rats. To understand the mechanistic basis for itstumorigenic activity, it is essential to elucidate the metabolic pathwaysof 2-NP in vivo. Such knowledge will also assist in developing analyticalmethods for monitoring human exposure to nitropolynuclear aromatichydrocarbons in ambient air.

Thus, 2-nitro|l/-4,5,9,10-14C]pyrene was synthesized and administered

to male F344 rats by intragastric gavage at a dose of 30 mg (0.4 mCi/mM)/kg body weight. During the first 48 h, 57.5% of the dose waseliminated in the feces and 9.7% was eliminated in the urine. Correspondingly, after 168 h, 58.9 and 10.6% were excreted in feces and urine,respectively. Fecal metabolites (isolated amounts) included 6-hydroxy-2-acetylaminopyrene (19.5%), 6-hydroxy-2-aminopyrene (10.4%), 2-ami-nopyrene (10.0%), 2-acetylaminopyrene (0.8%), and unmetabolized 2-nitropyrene (10.0%). 6-Hydroxy-2-acetylaminopyrene, 6-hydroxy-2-ami-nopyrene, and 2-aminopyrene were identified as their acetyl derivativesby comparison of their Chromatographie retention times, mass spectra,and IV spectra to those of synthetic standards. Urinary metabolitesincluded 6-hydroxy-2-acetylaminopyrene (2.0%); glucuronide conjugateswere tentatively identified (3.2%). The results of this study indicate thatnitroreduction and ring oxidation are metabolic pathways in vivo.

For DNA binding studies, rats were treated with 2-nitro|4,5,9,10-3H|

pyrene (1.6 mg (598 mCi/mM)/kg body weight]. The levels of binding(pM bound/mg DNA) were as follows: 1.3, liver; 1.14, mammary tissue;0.65, lung; 1.67, kidney; and 1.8, bladder. Upon high-performance liquidChromatographie analysis of the DNA hydrolysate (liver, mammary, andkidney), approximately 2.0% of the radioactivity coeluted with the synthetic markers derived from nitroreduction, ,V~(tU'o\yÂ«iianosin-H~yl)-2-aminopyrene and Ar-(deoxyadenosin-8-yl)-2-aminopyrene. Thus, simplenitroreduction of 2-NP does not significantly contribute to the total DNAbinding of 2-NP metabolites in vivo. The significance of each pathwayfor the tumorigenic effects of 2-NP remains to be examined.

INTRODUCTION

The mutagenic activities in bacterial and mammalian systemsand the tumorigenic activities in laboratory animals of severalmembers of the class of NO2-PAH4 have been clearly docu

mented (1). However, despite the widespread occurrence ofsuch agents in the environment, the risk that is associated withhuman exposure to NO2-PAH has not been clearly assessed (1-6).

In contrast to 1-NP, which is the most abundant NO2-PAHin environmental sources, its structural isomer 2-NP has beendetected only in ambient air and not in primary combustion

Received 7/22/91; accepted 12/17/91.The costs of publication of this article were defrayed in part by the payment

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

' This study was supported by National Cancer Institute Grant CA-35519.2 Present address: Food and Drug Administration, National Center for Toxi-

cological Research, Jefferson, AR 72079.'To whom requests for reprints should be addressed, at American Health

Foundation, 1 Dana Road, Valhalla, NY 10595.4The abbreviations used are: NO2-PAH, nitropolynuclear aromatic hydrocar

bons; 2-NP, 2-nitropyrene; 1-NP, 1-nitropyrene; 2-AP, 2-aminopyrene; 6-OH-2-NP, 6-hydroxy-2-nitropyrene; 6-OH-2-AP, 6-hydroxy-2-aminopyrene; 6-OH-2-AAP, 6-hydroxy-2-acetylaminopyrene; 6-Ac-2-AAP, 6-acetoxy-2-acetylamino-pyrene; 2-AAP, 2-acetylaminopyrene; N-dG-2-AP,JV-(deoxyguanosin-8-yl)-2-AP;N-dA-2-AP, iV-(deoxyadenosin-8-yl)-2-aminopyrene; N-dG-1-AP, /V-(deoxygu-anosin-8-yl)-l-aminopyrene; HPLC, high-performance liquid chromatography.

products such as diesel engine emissions (7-10). Extensivestudies have demonstrated the mutagenic and carcinogenicactivities of 1-NP; however, there is a paucity of informationon the biological activity of 2-NP despite its abundance inambient air, which is about equal to that of 1-NP (10,11). Both1-NP and 2-NP are direct-acting mutagens in several Salmo

nella tester strains, with the latter being much more active thanthe former (1,4, 12-14). While 2-NP requires nitroreductionfollowed by esterificai ion, 1-NP requires only nitroreductionfor expression of mutagenic activity. While both 1-NP and 2-NP yield a C8-deoxyguanosine adduct in Salmonella (14-16),a recent study demonstrated that 2-NP also yields a C8-deox-yadenosine adduct in relatively substantial amounts (14). Bothof these adducts were also identified as 2-NP-derived compounds in vitro (17,18). It appears that the position of the nitrogroup is an important factor in adduct distribution and induction of mutation in Salmonella strains.

Studies on the effect of the position of the nitro group ontumor induction are also scarce. Preliminary results indicatethat at 700 ^M/mouse, neither 1-NP nor 2-NP was active inthe newborn mouse assay.5 This is consistent with findings of

Wislocki et al. (19) and Busby et al. (20) who have demonstratedthat 1-NP at 80 ^M/mouse and 700 Â¿iM/mousewas ineffective;

only 2800 /Â¿M/mouseinduced liver tumors. In weanling femaleCD rats, after i.p. injection, only 2-NP induced leukemia/lymphoma(21); however, neither 1-NP nor 2-NP induced mammary tumors under the conditions of this study (total dose, 119Â¿Â¿M/rat;duration of the assay, 61 weeks). When the bioassaywas extended to 90 weeks, 1-NP induced mammary tumors;unfortunately, 2-NP was not tested under these conditions.These investigators concluded from this study that mammarytumor development by 1-NP in female Sprague-Dawley ratsrequired a long latency period. While the position of the nitrogroup appears to be critical for inducing mutation, its effect ontumor induction in laboratory animals needs further examination. Toward this goal and as an initial approach to bothmechanistic studies and possible dosimetry of exposure, weinvestigated the metabolism and DNA binding of 2-NP in vivoin the rat.

MATERIALS AND METHODS

Apparatus. HPLC was performed with a Waters Associates System(Millipore; Waters Division, Milford, MA) equipped with a Model 510solvent delivery system, a Model U6K septumless injector, a Model440 UV-visible detector, and a Model 680 Waters automated gradientcontroller. HPLC analyses were performed with a Vydac C18-10Â¿ireverse phase column (0.46 x 25-cm; Separations Group, Hesperia,CA) under the following conditions: System 1, a linear gradient from30% methanol/water to 100% methanol in 60 min at a flow rate of 1ml/min; System 2, a linear gradient from 30% methanol/water to 80%methanol/water in 30 min at a flow rate of 1.5 ml/min. Radioactivitywas measured with a Beckman Model LS9800 scintillation counter.For radiochromatography, we used a radioactive flow detector (Ra-diomatic Instruments and Chemical Co., Inc., Tampa, FL). Massspectra were obtained on a Hewlett-Packard Model HP5988A dual

'P. Upadhyaya, A. K. Roy, P. P. Fu, and K. El-Bayoumy, unpublished

observations.

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METABOLISM AND DNA BINDING OF 2-NP IN RATS

source mass spectrometer, and UV-visible spectra were recorded withan HP89530A spectrophotometer (Hewlett-Packard Co., Palo Alto,CA). 'H-Nuclear magnetic resonance spectra were run on a BrukerAM-360-MHz spectrophotometer in CDC3 (USA Bruker Instruments,Inc., Manning Park, Billerica, MA). Preparative thin-layer chromatog-raphy was done with silica gel 60 F254s-coatedplates (20 x 20 x 0.1 cm)with a preconcentration zone (4 x 20 cm; EM Science, Gibbstown, NJ).

Chemicals and Enzymes. Pyrene, 2,3-dichloro-5,6-dicyano-l,4-ben-zoquinonone, lead tetraacetate, and nitrobenzene were purchased fromAldrich Chemical Co., Inc., Milwaukee, WI, and 10% palladium onactivated carbon from Fluka, Switzerland. [4,5,9,10-uC]Pyrene (55mCi/mM, 95%) and [4,5,9,10-3H]2-NP (597 mCi/mM, 96%) were ac

quired from Chemsyn Science Laboratories, Lenexa, KS. Enzymes,purchased from Sigma Chemical Co., St. Louis, MO, included arylsul-fatase (from limpets, type V) and /3-glucuronidase (from Escherichiacoli, type IX), D-saccharic acid-l,4-lactone, alkaline phosphatase (typeIII from E. coli), phosphodiesterase I (type II from Crotalus adamanteusvenom), DNase I (type II from bovine pancreas), RNase A (type IAfrom bovine pancreas), RNase Tl (grade V from Aspergillus oryzal),and Proteinase K (type XI from Tritirachium album, fungal).

Synthesis of [uq2-NP. [l4C]Pyrene (166 mg, 6.25 mCi/mM) was

dissolved in 20 ml of ethyl acetate, and 200 mg of 10% palladium onactivated carbon was added to it. HydrogÃ©nation(55 i/-)was carried outat ambient temperature for 48 h in a Parr shaker (J. B. Thompson Co.)to yield [l4C]-4,5,9,10-tetrahydropyrene (144 mg, 87% yield). Its identity was confirmed by comparison of its 'H-nuclear magnetic resonanceand mass spectra with those of the unlabeled standard (22, 23). [14C]-

4,5,9,10-Tetrahydropyrene (144 mg, 6.25 mCi/mM) was dissolved in50 ml of acetic anhydride to which Cu(NO3)2-2.5 H2O (167 mg, 0.71HIM)was added with vigorous stirring. The reaction mixture was keptat room temperature overnight. After workup, ['4C]-2-nitro-4,5,9,10-

tetrahydropyrene was obtained and then purified (57.6 mg, 33%).Aromatization to [I4C]2-NP was achieved by refluxing [14C]2-nitro-

4,5,9,10-tetrahydropyrene (57.6 mg, 0.24 mM) with 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (214 mg, 0.94 mM) in 25 ml nitrobenzenefor 2 h as described (23). The crude product was purified in the HPLCSystem 1 to yield [14C]2-NP (retention time 55 min, 22 mg, 38.7%

yield).Synthesis of 2-AAP. The synthesis of 2-AP was described earlier

(17). 2-AP (4.5 mg, 0.02 mM) was dissolved in 20 ml of ethyl acetate,and acetic anhydride (0.5 ml) and 4-dimethylaminopyridine (1 mg)were added. After overnight stirring, the reaction mixture was washedwith a saturated solution of NaHCOj, and H2O. The ethyl acetateextract was dried over MgSO4. Pure 2-AAP was obtained by HPLC(System 1, retention time 44 min, 4 mg, 77% yield); MS (m/e, relativeintensity) 259 (M+, 68%), 217 (100%), 189 (50%). 'H-NMR (CDClj)&2.34 (s, 3H, CHj), 7.6 (s, IH, NH), 7.98 (t, IH, H7, J = 7.6 Hz), 8.02(d, 2H, H4+H,o or H5+H,, J = 9.0 Hz), 8.08 (d, 2H, H,+H, or H4+H10,J = 8.98 Hz), 8.18 (d, 2H, H6+H8, J = 7.65 Hz), and 8.34 (s, 2H,H,+H3)

Synthesis of 6-Ac-2-AAP. 6-OH-2-NP was synthesized as reportedpreviously (17). Excess Zn dust was added to a solution of 6-OH-2-NP(2.6 mg, 0.01 mM) in 5 ml of tetrahydrofuran containing 0.2 ml ofsaturated NH4C1 (24). The reaction was complete after 40 min asjudged by the disappearance of the starting material in HPLC System1. The reaction mixture was extracted with ethyl acetate after dilutingwith water. The ethyl acetate extract was dried (MgSO4), and the residuewas acetylated as described above and purified in HPLC System 1(retention time 36 min, approximately 1 mg, 43% yield); MS (m/e,relative intensity): 317 (M+ 20%), 275 (100%), 233 (50%), 204 (24%),176 (20%). 'H-NMR showed the following signals (CDC1,) &2.28 (s,

3H, OCOCHj), 2.58 (s, 3H, NHCOCHj), 7.60 (bs, IH, NH), 7.73 (d,IH, H7, J = 8.96 Hz), 7.98 (m, 4H, H4+H5+H,+H,0), 8.15 (d, IH, H8,J = 8.96 Hz), 8.17 (bs, IH, H, or H3), and 8.20 (bs, IH, H3 or H,)

Synthesis of 6-OH-2-AAP. 6-Ac-2-AAP (approximately 1.0 mg,0.003 mM) was dissolved in 3 ml of methanol, 100 mg of CH3ONa wasadded, and the mixture was stirred for 40 min, before acidifying withconcentrated HC1 and extraction with ethyl acetate. The pure productwas collected by HPLC in System 1 (retention time, 28-29 min); MS(m/e, relative intensity) 275 (M+, 90%), 233 (100%), and 204 (42%).

Synthesis of DNA Adduct Markers. N-dG-2-AP and N-dA-2-APwere synthesized as described previously (17, 18).

Metabolism of |'4q2-NP in Male F344 Rats. Male F344 rats (ap

proximately 250 g; Charles River Breeding Laboratories, Kingston,NY) were acclimatized to the conditions of the test laboratory for 2weeks before metabolism studies. Urine and feces were collected from3 rats, each housed in a glass metabolism cage (24). Rats received NIH-07 and water ad libitum. The metabolism cages were kept at 23 Â±1"C

and 50% humidity in light-controlled rooms (12 h light/dark cycle).Each rat was given [I4C]2-NP by intragastric gavage in trioctanoin at a

dose of 30 mg (0.4 mCi/mM)/kg body weight. The dose was selectedon the basis of metabolism studies with 1-nitropyrene in male F344rats. The excretion patterns and metabolite distributions were found tobe similar at doses of 30, 60, 80, and 100 mg of 1-NP/kg body weight.5Every 24 h for 6 days, urine was collected at 0-4Â°Cand feces werecollected at ambient temperature; both were stored below O'C until

analysis.Metabolism and DNA Binding of |'H|2-NP in Female Sprague-Dawley

Rats. Since female Sprague-Dawley rats are susceptible to tumor induction by nitrated pyrenes (21), the metabolism and DNA binding of2-NP were examined upon intragastric gavage in trioctanoin of [3H]2-

NP (1.6 mg/kg body weight; 598 mCi/mM) into 3 rats (approximately215 g; Charles River Breeding Laboratories), housed in metabolismcages. Only one sample each of urine and feces was collected 24 h after2-NP administration. The rats were sacrificed 24 h later; liver, lung,mammary fat pads, kidney, and bladder were isolated and kept at-78Â°Cbefore analysis.

Analysis of Urinary and Fecal Metabolites. Each 24 li urine samplewas extracted with ethyl acetate. The extracts were dried (MgSO4) andconcentrated to dryness. The residues were dissolved in tetrahydrofuranfor analysis in the HPLC System 1. Radioactivity in each urine samplewas measured before and after ethyl acetate extraction. Since we knewthat metabolites such as aminohydroxypyrenes are susceptible to decomposition (24), an aliquot of the ethyl acetate extract of the combinedurine samples was acetylated with acetic anhydride/4-dimethylamino-pyridine to ensure better recovery of such metabolites. The acetylatedmetabolites were analyzed in HPLC System 1.

To isolate water-soluble glucuronide and sulfate conjugates, aliquotsof urine samples that had been exhaustively extracted with ethyl acetatewere incubated in citrate buffer (pH 5.5) with /3-glucuronidase orarylsulfatase in the presence of saccharic acid-l,4-lactone at 37Â°Cfor 6

h. In a typical experiment, either 10,000 units of/3-glucuronidase or 63units of arylsulfatase and 20 mg of saccharic acid-l,4-lactone were usedfor each 10-ml pH-adjusted urine sample. Control experiments wereperformed as described above but without enzymes. The enzymaticreactions were quenched with 2 ml of ice-cold ethyl acetate and thereleased metabolites were further extracted with 4 x 10 ml of ethylacetate. The extract was dried and analyzed as such in the HPLCSystem 1, or it was further acetylated as described above and analyzedin the HPLC System 1.

Each 24-h fecal sample (8 g) was analyzed before and after acetylationas described for the analysis of urine. Another fecal sample (8 g) wassuspended in 50% ethyl acetate/ethanol and vortexed for 30 minfollowed by filtration; the residue was discarded. The filtrate wasconcentrated and redissolved in tetrahydrofuran, and radioactivity wasmeasured before HPLC analysis (System 1). Another part of the fecalsample (8 g) was suspended in 50% ethyl acetate/ethanol, and a mixtureof 1 ml acetic anhydride and 10 mg 4-dimethylamino-pyridine wasadded. This mixture was vortexed for 30 min and then stirred overnight.The residue was filtered; the filtrate containing the acetylated derivatives of metabolites was concentrated, and its radioactivity was measured and analyzed in the HPLC System 1.

Isolation and Analysis of 2-NP-DNA Adducts. Frozen livers (analyzedindividually) were thawed, minced, and then suspended in 0.01 M TrisHCl buffer, pH 7.4, containing 1% sodium dodecyl sulfate/1 mM EDTA(3 ml/g tissue) and homogenized with a Polytron. The homogenateswere incubated with proteinase K at 37Â°Cfor 40 min (0.5 mg/1 ml)

(25, 26). The mixture was extracted once with an equal volume ofphenol, then with a 1/1 mixture of phenol/Sevag (24/1 volumes ofchloroform/isoamyl alcohol), and finally with Sevag after adding NaCl

1177




(5 M, 0.05 volume) to the aqueous layer. DNA was precipitated byadding cold ethanol (2 volumes). The DNA was spooled out, washed 3times with 70% ethanol-water, dried, and redissolved in 2 ml of Trisbuffer (SO IHM.pH 7.4). Residual RNA was digested by incubation at37'C for 30 min with RNase Tl (50 units/ml) and RNase A (100 Mg/ml, preheated at 80'C for 15 min). The DNA was extracted as described

above, precipitated and redissolved in Tris buffer (pH 7.4), and thenquantified by its .-I;,,,,(1 .â€¢(.-,,â€ž= SO^g). Its purity was assessed by .I..,,,,/!.â€¢Â»"(1.8-1.9), and the levels of binding were calculated on the basis of

liquid scintillation counting. The yield of DNA was 2 mg/g liver. DNAfrom other organs was isolated as described for the liver, except thatmammary fat pads were first frozen in liquid N2 and ground to a powderin a stainless steel miniblender before being homogenized (26). DNAyields were 1.0 mg/g mammary tissue, 2.S mg/g lung, 2.6 mg/g kidney,and 0.3 mg/g bladder. The DNA was enzymatically hydrolyzed todeoxyribonucleosides (25, 26) followed by HPLC analysis in System 2.

RESULTS

The methods used for the synthesis of [MC]-2-NP were

adopted from the literature (22, 23). Starting from commercially available [MC]pyrene, the overall yield was 10.9%.

The time courses of excretion of radioactivity in urine andfeces after the p.o. administration of [14C]-2-NP to F344 rats

are shown in Fig. 1. During the first 48 h, 57.5% of theradioactivity was eliminated in the feces, and 9.7% in the urine.The corresponding values after 168 h were 58.9 and 10.6%,respectively. Fig. 2A shows a typical radiochromatogram obtained upon HPLC analysis of freshly isolated metabolites froma fecal sample, obtained 24 h after administration of 2-NP. Theindicated peaks were labeled, on the basis of their cochroma-tography with synthetic standards, as 6-OH-2-AP, 6-OH-2-AAP, 2-AP, 2-AAP, and 2-NP. Fecal metabolites were deriva-tized to corresponding acetyl derivatives to avoid decomposition and improve recovery of labile aminohydroxypyrene metabolites. Fig. IB shows a typical radiochromatogram of ace-tylated fecal metabolites. The indicated peaks were collectedfrom the HPLC, and their mass spectral data were identical tothose of synthetic standards (6-Ac-2-AAP and 2-AAP) as shownin Fig. 3. Unchanged 2-NP was also detected in feces. Thecombined fecal samples, collected over 6 days, were also analyzed by HPLC before and after acetylation, leading us toobserve no new metabolites by comparison to the sample obtained 24 h after 2-NP administration. Table 1 summarizes theresults obtained before acetylation. Under the experimentalconditions, 10.4% of the dose was excreted as 6-OH-2-AP,19.5% as 6-OH-2-AAP, 10% as 2-AP, 0.8% as 2-AAP, and10% as unchanged 2-NP. Levels of metabolites after acetylationwere comparable to those quantified before.

The ethyl acetate extracts of urine were analyzed by HPLC

25000T

20000-â€¢

[A]

48 72 98 120 144 168Time (Hours)

Fig. 1. Urinary and fecal excretion of radioactivity after administration of 2-nitro[4,5,9,10-'4C]pyrene to F344 rats by intragastric gavage.

E 15000--

10000-â€¢

5000-â€¢

io0

10180000-160000-

140000-120000-

100000-80000-60000-40000-20000

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0

10

Fig. 2. Radiochromatogram obtained upon HPLC analysis of the ethyl acetate/ethanol extract of feces from rats treated with 2-nitro[4,5,9,10-uC]pyrene, (A)before acetylation and (lÃ¬)after acetylation.

before acetylation (Fig. 4.4). On the basis of cochromatography,we ascertained the presence of 6-OH-2-AAP, 6-OH-2-AP, andseveral unidentified metabolites. Other standards (17) availableto us (e.g., 6-OH-2-NP) did not coelute with one of the majormetabolites eluting at 50-51 min, and there was insufficientmaterial for spectral analysis. 2-AP and 2-NP were not detected.The ethyl acetate extract of urine was also analyzed afteracetylation. Both 6-OH-2-AAP and 6-OH-2-AP were convertedto 6-AC-2-AAP (Fig. 4Ã„);the major peak that eluted at 50 minwas not affected by derivatization. Enzymatic hydrolysis ofwater-soluble metabolites revealed the presence of glucuronideconjugates of 6-OH-2-AAP; sulfate conjugates were not detected. The tentatively identified metabolites accounted forapproximately 5.7% of the dose or 53.8% of the radioactivityexcreted in the urine. These results are summarized in Table 1.

For the analysis of DNA binding, we used female Sprague-Dawley rats for reasons already described. Urine and feces werecollected and analyzed only after 24 h, and the rats weresacrificed thereafter. This time, we found 36% of the doseexcreted in feces and 4% in the urine over the course of 24 h.HPLC analysis of fecal and urinary metabolites established thepresence of the same metabolites identified above in male F344rats. However, the nature of water-soluble metabolites was notelucidated. DNA adducts formed in the liver, mammary glands,kidney, lung, and bladder of rats after the p.o. administrationof [4,5,9,10-3H]2-NP were isolated, enzymatically hydrolyzed,

and analyzed by HPLC. The levels of DNA binding in vivowere (pM bound/mg DNA, pool of 3 rats): 1.3, liver; 1.14,

1178




Fig. 3. Electron impact mass spectra of major metabolites obtained from rats treated with2-nitro[4,5,9,10-'4C]pyrene (bottom panels)

and those of standards (top panels).

100

80-

g 60H

Â» 40-1

20-

6 5

d", Â«1 1Â». ISO'}5

,nis""*

40 10 120 160 200 240â€”¿�iâ€”280 40 80 120 160 200 240 280 320 360

100-*"â€¢ÃŽ60-|

40-0)20-n-Isolated

metabolite *

afteracetylationiÂ»uÃ•

il,'i, , ,."!T7111*" Â»13!! 3S4 3I(

100-

80

60

40

20

o-V

Isolatedmetaboliteafteracetylation

i? m T

40 80 120 160 200 240 280 320 360 400 40 80 120 160 200 240 280 320 360 400m/e m/e

Table 1 Excretion of ("C]-2-nitropyrene and its metabolites in feces and urine

after intragastric garage to F344 rats% of dose isolated from":

Urine*Ethyl

acetate extract-able

after treatmentwith:Compound6-Hydroxy-2-acetylamino-

pyrene6-Hydroxy-2-aminopyrene2-Aminopyrene2-Acetylaminopyrene2-NitropyreneFecesf19.510.4

10.00.8

10.0Ethyl

acetateextractaMe**o.yND

NDND0-Glucu-

ronidase'NDND

NDNDAryl-

sulfataseNDÂ«ND

NDNDND

" Isolated amounts unconnected for possible losses in analysis; each value was

obtained from pooled urine or feces from 3 animals.* 10.6% of the dose was excreted in the urine.' 58.9% of the dose was extracted from the feces.**6.0% of the dose was extracted from the urine with ethyl acetate.' 6.7% of the dose was extracted from urine after treatment with /3-glucuroni-

dase.' Identification was based on cochromatography.* ND, not detected.

mammary tissue; 0.65, lung; 1.67, kidney; and 1.8, bladder.Upon HPLC analysis of the liver DNA hydrolysates, the bulkof the radioactivity eluted in the void volume and in the meth-anol wash. As shown in Fig. 5, approximately 2% of theradioactivity was eluted with a synthetic mixture of N-dG-2-AP and N-dA-2-AP (17, 18). Similar results were obtainedupon analysis of DNA hydrolysates obtained from mammaryglands, lung, and kidney.

DISCUSSION

Excretion of 2-NP and metabolites occurs mainly via thegastrointestinal tract. The pattern of excretion is similar to thatreported previously in the metabolism of its most extensively

studied structural isomer, 1-NP (18, 24, 27). We have shownthat 51% of the dose was excreted in the feces and 19% in theurine 5 days after intragastric gavage of 1-NP (24). Marshall etal. (28) found that 70% of a p.o. administered dose of 1-NPwas excreted in the feces. Ball et al. (29) have also recoveredmore than 70% of the dose in the feces after an i.p. injection of1-NP to either male AGUS or male Sprague-Dawley rats (29,30). However, when rats were exposed to 1-NP in the form ofan aerosol, urine was the major route of excretion (31 ). Takentogether, all of these findings indicate that the route of administration has great influence on the pattern of excretion of NO2-PAH. Since human exposure to 2-NP is probably occurring byinhalation, future investigations should focus on inhalationexperiments. This is especially important with regard to developing markers for human exposure to 2-NP in ambient air.

The present investigation shows that 2-NP can be metabolized in vivo in the rat via both nitroreduction and ring oxidation. In vitro metabolism of 2-NP using 9000 x g rat liversupernatant also yielded metabolites derived from both pathways (17). Numerous other studies in vivo and in vitro have alsoshown that other NO2-PAH can be metabolically activated bynitroreduction and ring oxidation (6). We found this to be so,not only in adult animals but also in newborn animals (25).Metabolic reduction of 2-NP to 2-AP suggests the formationin vivo of the active 2-hydroxylaminopyrene, which has beenshown to react with DNA in vitro to yield N-dG-2-AP and N-dA-2-AP (18). Xanthine oxidase-catalyzed formation of 2-hydroxylaminopyrene from 2-NP in vitro in the presence of DNAresulted in the same 2 adducts (17). Rat and mouse livermicrosomes or cytosols metabolize 2-NP in the presence ofDNA to both adducts presumably via the hydroxylamine (18),suggesting that nitroreduction may contribute to the metabolicactivation of 2-NP to a tumorigen (21). The identification of 6-OH-2-AP as a metabolite points to the fact that the formationof hydroxylamines other than that derived from the parentcompound 2-NP is also plausible. Such putative intermediates

1179




30000T

25000' â€¢¿�

20000- â€¢¿�

15000"

10000- â€¢¿�

5000-

200000

160000-

120000-

80000-

40000

MIN

Fig. 4. Radiochromatogram obtained upon HIM < analysis of the ethyl acetateextract of urine from rats treated with 2-nitro[4,5,9,10-'4C]pyrene, (A) before

acetylation and (B) after acetylation.

could contribute to the overall metabolic activation of 2-NP invivo. Ring-hydroxylated metabolites of 1-NP have also been

shown to react with DNA in vitro, and in some instances theywere more mutagenic than the parent compound 1-NP (25,32).These observations further strengthen the evidence for activation of NO2-PAH by both ring oxidation and nitroreductionpathways.

By analogy to 1-NP (29, 30, 33), a glucuronic acid derivativeof 6-OH-2-AAP constitutes the major conjugate detected in theurine after 2-NP administration. In contrast to 1-NP, sulfateconjugates were not detected in the present study. Since 6-OH-2-AAP and its conjugate are prevalent metabolites of 2-NP, itis tempting to propose that such metabolites can be utilized asan indicator for monitoring human exposure to 2-NP in ourenvironment. Glutathione conjugates derived from ring-oxidized metabolites of 1-NP, such as 4,5-epoxy-4,5-dihydro-l-nitropyrene and 9,10-epoxy-9,10-dihydro-l-nitropyrene, havebeen identified (34, 35), and similar conjugates may accountfor the unknown metabolites detected in the urine (5.1%; Table1) of rats treated with 2-NP. This deserves further examination.In addition, the role of the identified metabolites in this studyin inducing mutation and tumorigenesis remains to be elucidated (14, 21).

We also examined the DNA binding of 2-NP in the liver,mammary' tissues, lung, kidney, and bladder tissues to deter

mine the potential role of nitroreduction. Thus, adducts derivedfrom nitroreduction that have been identified in vitro (17, 18)

20 30MIN

Fig. 5. Radiochromatogram obtained upon HPLC analysis of liver DNAhydrolysate from rats treated with [4,5,9,10-3H]-2-nitropyrene. Arrow, retentiontime of /V-(deoxyguanosin-8-yl)-2-aminopyrene and /V-(deoxyadenosin-8-yl)-2-aminopyrene.

were used as markers in the in vivo study. Appreciable amountsof radioactivity were bound to DNA in the organs and tissuesexamined. However, upon HPLC analysis of the DNA hydrol-yzates (liver, breast, and kidney), approximately 2% eluted withthe standard mixture of N-dG-2-AP and N-dA-2-AP adducts.On the basis of cochromatography, it appears as if these DNAadducts, which are derived from nitroreduction, are formed invivo. The relative amount of each adduct cannot be determineddue to the low levels of radioactivity and only very smalldifferences in retention times between the 2 adducts (17, 18).However, it needs to be emphasized that such adducts appearto be important in mutation induction by 2-NP in Salmonellatyphimurium (14). Studies in our laboratory and elsewhere havedemonstrated the presence of multiple adducts including thatderived from nitroreduction of 1-NP in vÃ¬vo,N-dG-1-AP (Ref.36 and references therein). This can be a major or a minoradduct depending on the experimental protocol (36). The importance of nitroreduction in mutation induction by 1-NP and2-NP has been demonstrated (14-16). The role of nitroreduction in tumor induction by 1-NP has not been clearly defined,and in the case of 2-NP it needs to be fully investigated.

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1992;52:1176-1181. Cancer Res Pramod Upadhyaya, Ajit K. Roy, Peter P. Fu, et al. Metabolism and DNA Binding of 2-Nitropyrene in the Rat

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