Metabolism of 1-nitropyrene and formation of DNA

Carclnogenesis, Volume 2 Number 10 1981 p.1007-1011

Metabolism of 1-nitropyrene and formation of DNA adducts in Salmonella typhimurium F. Messier1, C. Lu2, P. Andrews1, B.E. McCarry1, M.A. Quilltam1 and D.R. McCalla2 3 Departments of Chemistry 1 and Biochemistry2, McMaster University, Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.

Abstract 1-Nitropyrene is slowly reduced by intact cells of Salmonella typhimurium to yield 1-aminopyrene and N-acetyH-aminopyrene phis six unidentified minor products. When the bacteria are exposed to tritiated 1-aitropyrene, increasing amounts of radioactivity become bound to DNA as the nitropyrene is metabolized. Enzymatic hydrolysis of the labelled DNA yields low molecular weight labelled compounds which probably represent nudeoside adducts formed by the reaction of nitropyrene metabolites with DNA. Results with appropriate mutant strains indicate that bacterial nitroreductases are involved in activating nitropyrene to a reactive intermediate that binds to DNA and that nitropyrene adducts in DNA are subject to excision repair. Introduction Since the work of Pitts (1) there has been increasing interest in the possible environmental hazards posed by nitro derivatives of polycyclic aromatic hydrocarbons (nitro-PAHs)*. More recently several nitropyrenes were found as trace impurities in carbon black used in older batches of xerographic photocopier toners (2,3). NitroPAHs probably account for much of the mutagenic activity of particulate material from diesel exhaust (4,5). In addition, some nitro-PAHs, (e.g., 2,4,7-trinitrofluorene-9-one), are used as photosensitizers in other photocopying processes (6). Nitro-PAHs as a class are powerful direct acting mutagens in the Salmonella assay (1,3). From the limited results available, it appears that these compounds are also mutagenic to mammalian cells but that they are more potent in Salmonella (6,7). The work of Rosenkranz and his collaborators (3,8) has indicated that the nitropyrenes, like nitroheterocyclic compounds, are probably "activated" by reduction by endogenous bacterial nitroreductases since mutants lacking these enzymes are partially resistant to these compounds. In this paper we report that 1-nitropyrene is slowly 'Author to whom reprint requests should be addressed. •Abbreviation: nitro-PAHs, nitro derivatives of polycyclic aromatic hydrocarbons.

©

Experimental Synthesis 1-Nitropyrene was prepared by treating pyrene with nitric acid in acetic anhydride at 25 °C (9) and purified by preparative reverse phase h.p.l.c. on a Magnum-9 ODS-II (0.9 x 50 cm) column. 1 -Aminopyrene was synthesized by hydrogenating a sample of purified 1-nitropyrene at atmospheric pressure over Adam's catalyst in methanol. The acetylation of 1-aminopyrene in dichloromethane with acetic anhydride yielded N-acetyl-1-aminopyrene. H.p.l.c. analyses of 1-aminopyrene and its N-acetyl derivative showed a single, unique peak for each product. Mass spectrometric analysis of these products confirmed their identity. Tritiated 1-nitropyrene, synthesized by the nitration of tritiated pyrene, was purified on an analytical h.p.l.c. column (Merck RP-18, 4,6 x 250 mm; solvent, methanol: acetonitrile: water, 70:30:10) to afford products with specific activities of 12.6 and 16.4 Ci/mmol in two preparations. The tritiated pyrene was prepared by the hydrogenolysis of 1-bromopyrene (10) with tritium gas (reduction performed by Amersham Corp., Oakville, Ontario, Canada). Liquid chromatography H.p.l.c. of nitropyrene metabolites was performed on a Spectra-Physics Model SP-8000 instrument. An Altex reverse phase 5 /im Ultrasphere-ODS column (250 mm x 4.6 mm I.D.) was used with gradient elution from 60% aqueous methanol to 100% methanol over 50 min. The eluent was monitored at 254 nm with a Beckman Model 154 u.v. absorption detector. An Isco Model 328 fraction collector was used for collecting fractions directly into scintillation vials for determination of tritium. Fractions recovered for subsequent mass spectral analysis were collected in glass tubes and immediately evaporated to dryness with a Savant Speed-Vac concentrator. The residue was then transferred with 20 id of dichloromethane into melting point capillaries and the solvent again evaporated. These tubes were sealed and submitted for mass spectral analysis. Mass spectrometry All mass spectra were acquired on a VG Micromass 7070F mass spectrometer with an electron energy of 70

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1007

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(Received on 2 June 1981; accepted on 17 July 1981)

metabolized by intact cells of Salmonella typhimurium, to yield 1-aminopyrene and N-acetyl-1-aminopyrene. We further report that when tritiated nitropyrene is metabolized, some radioactivity becomes bound to DNA. After the enzymatic hydrolysis of such labelled DNA, the radioactivity is found in low molecular weight material. In nitroreductase-deficient Salmonella much less radioactivity becomes bound to DNA.

F. Messier, et al.

Metabolism and DNA adducts An overnight culture of bacteria (150 ml) was diluted to 11 and incubated at 37°C with forced aeration. When the AJQO of the culture reached 0.60 the culture was divided into several 150 ml samples and 30 /*g of 1-nitropyrene in 75 y\ dimethylsulfoxide added to each. The cultures were then incubated at 37°C in a shaking water bath. The cell density doubled during the first 2 h and thereafter remained essentially constant. At intervals, the cells were removed by centrifugation at 10,000 x g and the supernatant extracted three times with 40 ml of CHJC\2. This procedure extracted over 90% of the radioactivity. Benzo[a]pyrene (70 ng) was added as an internal standard, the CH2C12 evaporated and the residue transferred to a small vial with CH2C12 (2 ml) which was then evaporated under a stream of nitrogen. Forh.p.l.c. analysis the residue was dissolved in 70 jd of MeOH and 10 /xl aliquots injected. For the DNA binding experiments, 30/ig (0.12 jtmol; 9.2 x 107 d.p.m.) of [3H]nitropyrene in 100 /tl of methanol was used for each 150 ml culture. After appropriate incubation at 37°C with shaking, the cells were removed and the DNA prepared by phenol extraction as previously described (14). In order to assess the extent of metabolism of 1-nitropyrene in these experiments, the supernatant after harvesting the bacteria was extracted as described earlier (except that internal standard was not added) and analyzed by h.p.l.c. Fractions were collected in scintillation vials and analyzed for tritium in a Beckman LS230 scintillation counter. Hydrolysis of DNA DNA was treated successively with DNase I, snake venom phosphodiesterase and alkaline phosphatase as described by King et al. (15). Results Figure 1 shows the h.p.l.c. profiles for u.v. absorbing material and radioactivity in extracts of the supernatant from a culture of S. typhimurium TA98 which had been allowed to metabolize [3H]nitropyrene for 24 h. While some nitropyrene remained unchanged, two major and 1008

18

24 Minutes

Fig. 1. Metabolism of 1-nitropyrene by S. typhimurium TA98. Bacteria were incubated with 30 /ig (9.2 x 10' d.p.m. [3H]nitropyrene in 150 ml of defined medium for 24 h after which a dichloromethane extract of the medium was chromatographed on a reverse phase Cu column. Top panel: absorbance at 254ran;lower panel: radioactivity. Peak identities: 4 = N-acetyl-1-aminopyrene; 6 = 1-aminopyrene; 8 = 1-nitropyrene. Relative peak area from radiochromatogram: 1 = 2%; 2 = 0.8%, 3 = 0.2%; 4 = 20.5%; 5 = 0.2%; 6 = 70.6%; 7 = 1.0%; 8 = 4.4%; 9 = 0.3%.

six minor labelled metabolites were also present. The major components have been identified as 1-aminopyrene (21 min) and N-acetyl-1-aminopyrene (15.5 min) on the basis of their h.p.l.c. retention times and mass spectra (Figure 2) matching with those of authentic standards. The u.v. absorbing material which eluted before 6 min was also found in control experiments in which no nitropyrene was added. Figure 3 shows the time-course for the reduction of 1-nitropyrene by intact cells of S. typhimurium and the appearance of 1-aminopyrene and N-acetyl-1aminopyrene. These data are typical of those obtained in several consecutive experiments. Reduction of nitropyrene was very much slower than the reduction of nitrofurazone, a nitrofuran derivative, under the same conditions. The binding data (Table I) are reported as d.p.m./lOO /tg DNA to avoid problems caused by variable recoveries of DNA. The extent of binding of radioactivity from 1-nitropyrene to DNA in strain TA98 increased with the time of incubation (experiment 3) and was dependent on the amount of mutagen available per cell (experiment 4). The maximum levels of binding were of the order of one molecule of nitropyrene per 10* nucleotides in DNA. When DNA isolated from strain TA98 was chromatographed on a 60 x 1.5 cm diameter column of Sephadex G25, the u.v. absorption and


eV and a source temperature of 200°C. Samples were introduced via the solid probe inlet. Bacteria Four strains of S. typhimurium kindly provided by Dr. B.N. Ames, Berkeley.CA, USA, were used (11): TA98 (his D3052, A uvr B rfa plus pKM 101 plasmid; TA100 his G46, A uvr B rfa plus pKM 101 plasmid; TA1538 his D3052, A uvr B rfa and TA1978 the uvr* analogue of TA1538 (11). TA100 F50, a nitrofuranresistant mutant which is partially deficient in nitroreductase activity (12), was obtained from Dr. H.S. Rosenkranz, New York, NY, USA. Cultures were maintained on nutrient agar and grown in Davis — Mingioli medium (13) supplemented with glucose, histidine and bio tin.

1-Nltropyrcne metabolism and DNA adducts In S. typhimurium

Table I Binding of ['HJnitropyrene metabolites to DNA in S. typhimurium under standard conditions. Experiment no. 1

Strain

Time(h)

D.p.m./lOOfig DNA

S. typhimurium S. typhimurium S. typhimurium

TA98 TA98 TA98 •

16 16 2 5 16 16 16 16 16 16 16 16 16

7340 8210 1560 2990 7480 4260 1100 2920 670 2340 690 1870 648

m m

m

TA98

S. typhimurium '

b

S. typhimurium

m

TA1538 TA1978 TA100 TA100 F50 TA100 TA100 F50

m

S. typhimurium m

S. typhimurium m

See text for details. "Experiments 1 —4 were done with 1-nitropyrene having a specific activity of 16.4 Ci/mmol; in experiment 5 — 7 the specific activity was 12.6 Ci/mmol. b In this experiment the cells were four times as concentrated as usual so that the dose per cell was 1/4 of that in the other treatments.

. i J. I

NHCOCH3 Mt 259

Fig. 2. Electron-impact mass spectra of metabolites of 1-nitropyrene isolated by liquid chromatography. Top panel: l-aminopyrene (peak 6 in Fig. 1); lower panel: N-acetyl-l-aminopyrene (peak 4 in Fig. 1).

radioactivity profiles coincided (Figure 4). Upon enzymatic hydrolysis of the DNA, the radioactivity appeared in low molecular weight material, much of which eluted before the normal nucleosides.

Fig. 3. Conversion of 1-nitropyrene to l-aminopyrene and N-acetyl1-aminopyrene by S. typhimurium TA98. The vertical scale shows, for the pyrene derivatives, the mol percent relative to the initial 1-nitropyrene level (obtained from h.p.l.c. peak areas corrected for 254 nm extinction coefficients). Data for the disappearance (measured as the AAJJJ) of nitrofurazone (NOrfurazone) is also shown. The initial concentration of nitrofurazone was 0.8 jiM, identical to that of 1-nitropyrene. Other conditions were also identical. Data are expressed as the percentage of nitrofurazone remaining at various times.

Strain TA100 F50 is a mutant of TA100 which was selected on the basis of resistance to nitrofurazone (12) and is known to be partially lacking in nitroreductase activity which is required for the activation of nitrofurans. In this strain, only about one third as much radioactivity became bound to DNA as in TA100 (Table I, experiments 6 and 7). In a separate experiment, the amount of labelled 1 -nitropyrene in the supernatant was measured after 16 h incubation. In the culture medium from TA100, 23% of the radioactivity recovered was 1009


1 2 3a 3b 3c 4a 4b 5a 5b 6a 6b 7a 7b

Species

F. Messier, et al.

found in 1-nitropyrene while in the F50 mutant, 50% of the label remained in 1-nitropyrene. This indicates that the mutant is partially lacking in 1-nitropyrene reductase activity. Less radioactivity from nitropyrene was found in association with the DNA of a repair-proficient strain (TA1978) than in strain TA1538, an otherwise isogenic repair-proficient strain (Table I, experiment 5). This difference is presumably a consequence of the removal of some of the labelled adducts that were formed in the repair-proficient strain. Discussion These results provide direct confirmation of the hypothesis developed by Rosenkranz et al. (3) on the basis of mutational studies. Firstly, cells of S. typhimurium do reduce nitropyrene to yield corresponding amino and acetylamino derivatives. However, the rate of reduction of nitropyrene is very slow compared to that of a nitrofuran derivative at the same molar concentration — a fact which may complicate studies of nitro-PAHs reduction in the cell free extracts. Secondly, radioactivity from [3H]nitropyrene becomes bound to bacterial DNA with a time-course similar to that for the reduction of 1-nitropyrene. Many aromatic amines, including 1-aminopyrene, are mutagenic to S. typhimurium after oxidative metabolism by S-9 preparations (16), whereas nitropyrene is "direct acting" (in the sense that no external activation system is required). From the data reported here, and by Rosenkranz et al. (3) and Mermelstein et al. (17), plus what is known about the action of nitroheterocyclic compounds (18), it seems most likely that nitropyrene is reduced by the bacteria to give a highly reactive species which can either react with DNA (and other cellular constituents) or become fur1010

Acknowledgemen ts This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). Dr. F. Messier was the recipient of an NSERC postdoctoral fellowship. We thank Drs. R. Mermelstein and H.S. Rosenkranz for helpful discussions.

References 1. Pitts.J.N. Jr., Van Cauwenberghe.K.A., Grosjean.D., Schmid, J.P., Fitz.D.R., Belser.W.L. Jr., Knudson.G.B., and Hynds.P.M. (1978), Atmospheric reactions of polycyclic aromatic hydrocarbons: facile formation of mutagenic nitro derivatives, Science (Wash.), 202, 515-518. 2. Lofroth.G., Hefner.E., Alfheim,I., and MoUer.M. (1980), Mutagenic activity in photocopies, Science (Wash.), 209, 1037-1039. 3. Rosenkranz.H.S., McCoy,E,C, Sanders.D.R., Butler^C., Kiriazides.D.K., and Mermelstein,R. (1980), Nitropyrenes: isolation, identification and reduction of mutagenic impurities in carbon black and toners. Science (Wash.), 209, 1039-1043. 4. Shuetzle.D., Prater.T.J., Riky.T., Durisin^A., and Salmeen, I. (1980), Analysis of nitrated derivatives of PAH and determination of their contribution to Ames assay mutagenicity for diesel paniculate extracts, 5th International Symposium Polynudear Aromatic Hydrocarbons, Columbus, OH, Abstracts.


Fig. 4. Chromatography on Sephadex G25 of DNA isolated from S. typhimurium after exposure to [^Jnitropyrene for 16 h. Top panel: DNA before hydrolysis; lower panel: products of enzymatic hydrolysis of the DNA. See text for details.

ther reduced to the amine. It is of interest that Rosenkranz et al. have presented preliminary evidence indicating that reduction of 1,8-dinitropyrene with zinc and ammonium chloride generated an unidentified compound which was much more mutagenic to a mutant lacking nitroreductase than was the original nitro compound (17). The results of the DNA binding experiments with the nitroreductase-deficient strain, TA100 F50, are of interest and imply that the reductase activity which is present in TA100 (wild-type for nitroreductase) but absent from the F50 mutant is involved in the reductive "activation" of 1-nitropyrene. This result is again consistent with the results of Mermelstein et al. (17) who reported that a mutant of TA98 selected in the same way as TA100 F50, (i.e., on the basis of resistance to nitrofurazone), was resistant to the mutagenic effect of nitropyrene. It is not dear why the binding of radioactivity from 1-nitropyrene was consistently greater in TA98 than in the other strains. The results of Figure 4 show that the radioactivity found in preparations of DNA from bacteria exposed to [3H]nitropyrene was very closely associated with DNA. After hydrolysis of the labelled DNA, no tritium was found in the DNA region of the chromatogram but, rather, the tritium eluted in a well-defined and lower molecular weight zone and was partially separated from the normal nucleosides. While other interpretations are possible, it seems most likely that the material ehiting at 24 -L 34 ml in Figure 4 (lower panel) represents an adduct or adducts formed between nitropyrene metabolites and nucleosides in DNA. From our results with the uvr* strain and from the mutational data of Rosenkranz et al. (17), it appears that these adducts are subject to removal in strains proficient in excision repair. Further work on the isolation and characterization of these compounds is in progress.

1-NItropvrene metabolism and DNA adducti in 5. typhimurium

microsome mutagenicity test, Mutat. Res., 31, 347-364. 12. Rosenkranz.H.S., and Speck.W.T. (1976) Activation of nitrofurantoin to a mutagen by rat liver nitroreductase, Biochem. Pharmacol., 25, 1555-1556. 13. Davis.B.D., and Mingioli.E.S. (1950), Mutants of Escherichia coli requiring methionine or vitamin B u , J. Bacteriol., 60, 17-28. 14. Wentzell.B., and McCalla.D.R. (1980), Formation and excision of nitrofuran-DNA adducts in Escherichia coli, Chem.-Biol. Interactions, 31, 133-150. 15. King.H.W.S., Thompson.M.H., and Brookes.P. (1975), The benzo[a]pyrene deoxyribonucleoside products isolated from DNA after metabolism of benzo[a]pyrene by rat liver microsomes in the presence of DNA, Cancer Res., 35, 1263-1269. 16. McCann.J., Choi.E., Yamasaki.E., and Ames.B.N. (1975), Detection of carcinogens as mutagens in the So//wcwie//a/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. USA, 72, 51355139. 17. Mermelstein.E., Rosenkranz.H.S., and McCoy.E.C. (1981), The microbial mutagenicity of nitroarenes, A UI/BNL Symposium on the Genotoxic Effects of Airborne Agents. 18. McCalla.D.R. (1979), Nitrofurans, in Hahan.F.E. (ed.), Antibiotics V-l. Mechanism of Action of Antibacterial Agents, SpringerVerlag, New York, 1979.

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5. Schuetzle,D., Lee,F.S.-C., and Prater.T.J. (1981), The identification of polynuclear aromatic hydrocarbons (PAH) derivatives in mutagenic fractions of diesel particulate extracts, Int. J. Environ. Anal. Chan., 9, 93-144. 6. Burrdl.A.D., AndeTson.J.J., Jotz,M.M., Evans.E.L., and Mitchell ,A.D. (1981), Genetic toxkity of 2,4,7-trinitrofluorene-9-one in the Salmonella assay, L5178 TK+/' mouse lymphoma cell mutagenesis assay and sister chromatid exchange assay, Abstracts of the Environmental Mutagen Society, 12, 121-122. 7. Arlett.C.F., Cole.J., Broughton.B.C, Laine.J., and Bridges,B.A. (1981), Mutagenic effects in human and mouse cells by a nitropyrene, A UI/BNL Symposium on the Genotoxic Effects of Airborne Agents. 8. Rosenkranz.H.S., McCoy.E.C, Mermelstein.R., and Speck.W.T. (1981), A cautionary note on the use of nitroreductasedeficient strains of Salmonella typhimurium for the detection of nitroarenes as mutagens in complex mixtures including diesel exhausts, Mutat. Res., 91,103-105. 9. Bavin.PJvi.G., and Dewar.M.J.S. (1956), Electrophilic substitution, Part I: Preliminary investigations, / . Chan. Soc., 164-169. 10. Lock.G. (1937), Uber abkommHnge des Pyrens, Chem. Ber., 70, 926-930. 11. Ames.B.N., McCann.J., and Yamasaki.E. (1975), Methods for detecting carcinogens and mutagens with the &/mone//a/mammalian