The extraordinary mutagenicity of nitropyrenes in bacteria

The extraordinary mutagenicity of nitropyrenes in bacteria

187 Mutation Research, 89 (1981) 3 87-196 E~s~vi~r/N~rth-Holland Biamed!caf Press Nitropyrenes cause frameshift mutations in SaZmonelEa typhimurium...

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187

Mutation Research, 89 (1981) 3 87-196 E~s~vi~r/N~rth-Holland Biamed!caf Press

Nitropyrenes cause frameshift mutations in SaZmonelEa typhimurium. This activity which is restricted to frameshift mutations is unusual in several respects: (a) Nitropyrenes, as a class, are the most mutagenic chemicals reported in the litera~re; (b) The mutagenicity depends upon the formation of adducts between DNA and nitropyrene metabaiites; (c) The penultimate intermediates responsible for mutagenic activity (bydroxy~m~nes~ are not obtained in all instances by reduction sf the nitro function by the ‘*classical” ~i~oreductase (the one that acts on nitrofurans and other simple nitrated polycyclic aromatic hydr~c~b~ns~ but by another ni~oreduc~se which appears to be specific for higher nitrated polycychc aromatic hydroc~bons~ (d) The mutagenicity of nitropyrenes is enhanced when resting rather than growing bacterial cultures are used.

A recent &udy from these ~ab~ra~~ries described the is&&ion, identification and reduction of mutagenic nitropyrene trace impurities in a carbon black and selected ~@ro~aph~c toners (Ro~n~anz et al., 1380a). ~i~o~enes have been detected in various en~o~rne~~ samples (Pitch et al., 1978; Pitch and Smith. 1979; &ger, 1978; Pitts et al., 1975; Pitts, 1979; Wig et al., 1978; Wei et al,, 1980; Kubitsch~k and W~liams, 1980; Lofroth, 1980; King et al., 1980). These compounds presumably result from the ambient nitration of polycyclic aromatic hydrocarbons which originate from incomplete mobile and stationary combustion processes. Nitroarenes are present in extracts of diesel exhaust particulates and among these I-nitropyrene is responsible for a subPontiac portion of the observed d~ect~~ting mu~genici~ activity in the Salmonella assay (Pederson and Siak, 1980; Tokiwa et al., 1979; Schuetzle et al., 1980, 1981). The present reps& is conceded with a more detailed description of the rnu~ge~c prop~ies of six nitrated derivatives of pyrene.

188

aterials and methods Nitropyr~~~s were synthesized as described previously ~Rosenkra~z et al., 1980a) with tne exception of ~,3,6~S-tetranitropyren~, they were purified by HPLC and they are believed to be at least 99.0% pure. The Salmonella tester strains TA1535, TAlOO, TA1537, TA1538, TA98, TA2978 and TA1977 were obtained from Dr. R.N. Ames, University of California, Berkeley. E. coli WPBEWA was obtained from Dr. Michael Green, MRC Cell Mu~tion Unit, University of Sussex. Salmonella typhimurium TA98NR is a derivative of TA98 which is deficient in nitroreductase. It was isolated as a nitrofuran-resi,stant mutant (Rosenkranz and Speck, 1975). The retention of the deep-rough character and of the plasmid pkMlOl was ascertained {Ames et al., 1975). Salmonella mu~geni~ity assays were carried out as described by Ames et al. (1975) except that unless indicated otherwise, exponentially growing cultures were used. The E. cdi mutagenicity assay was that described by Green and Muriel (1976). Every dose level was run in triplicate and every chemical was run on at least 3 separate occasions. In addition, assays with strains TA98 and TA98NR were carried out on at least 10 separate occasions. Results and discussion All 6 of the nitropyrenes investigated exhibited strong mutagenic activity for s~~~o~e~~~ ~y~~j~~riu~ in the absence of mi~rosom~ activation mixtures. Addition of such activation mixtures actually resulted in a substantial decrease in the mutagenicity of nitropyrenes (Rosenkranz et al., 198Oa). The potent mutagenic activity of nitropyrenes in tester strains TA98, TA1538 and TA1537 is indicative frameshift mutations. In contrast generally there was no activity observed with strains TAl.535 and E. coli ~P2~u~A which are used to detect base-substitution mutations. The activity exhibited in TA108 presumably reflects the decreased mutagenic specificity of this tester &rain resulting from the acquisition of the plasmid pKM101 (McCann et al., 1975). Unexpectedly, the mutagenic activity of three of the nitropyrenes was not decreased when they were tested in strain TA98NR, the nitroreduct~edeficient derivative of TA98 that has been shown to lack the enzyme which activates nitrofurans, nitronaphthalenes, and nitrofluorenes to mutagens (Rosen.kranz and Speck, 1975; Rosenkranz and Poirier 1979; Rosenkranz and Mermelstein, 1980). These results suggest that the nitropyrenes are either direct-acting and do not require the usual reductive conversion to the ~o~esponding hydroxylamines, or if a reductive process does occur that it is performed by an enzyme -her than the “classical” nitroi-eductase. A plausible mechanism for the obved direct frameshift activity could be due to intercalation of these chemi4 between DNA base pairs. There are, however, 3 types of evidence which ii re against such a mechanism. First, non-adduct-forming mutagens are usually active p~,cip~ly in strain TA1537 rather than TA1538 and its derivatives I.4mes et al., 1973; Speck and Rosenkranz, 1980, Rosenkranz and Mermelstem, 1980). This is not the case with the nitropyrenes. In addition, physical-chemical studies of mixtures of DNA and nitropyrenes did not indi-

f,B-DINITROPYRENE

SCHEME

:

cate such intercalative interactions (i.e. no spectral shifts, no increases in the temperature of the thermal helix-to-coil transition profile, no effect on the sedimentation rate of closed circular DNA; unpublished results). Finally, it was demonstrated that the mutagenic activity of nitropyrenes was due to adduct formation as indicated by t.he decreased mutagenicity seen with tester strain TA1978 and TAf977 which are UWB’ analogs of TA1533 and TA1537, respectively. It has been shown, that while these strains respond in an undiminished fashion to chemicals which cause frameshift mutation as a result of intercalation between DNA-base pairs, they show a greatly diminished mutagenic response when exposed to chemicals which form adducts that are recognized by UV endonuclease, the product of the uurB+ gene (Ames, 1972; Ames et al., 1973; Rosenkranz and Mermelstein, 1980). One possible hypothesis to explain the direct reaction of nitropyrenes .with DNA would involve an analogy with the well documented reaction of benz[a]pyrene diolepoxide with cellular DNA (Jerina et al., 1978). In the latter case (Fig. l), opening of the epoxide results in the f~jrmation of a benzylie, resonate-stab~ized* c~boni~m ion, which is capable of birding to the exocy clic amino group of d~xy~anosine. In the case of the ~it~o~yrenes, a resonance-stabilized nitronium may be formed which could then bind to cellul~ DNA. Such a scheme would account for the observed mutagenicity pattern of the nitropyrenes and obviate the need for the reduction of the nitro moiety to the penultimate hydroxylamine whose esterification may result in a potent electrophilic reagent (Miller, 1970). Elowever, no physical chemical change in DNA exposed in vitro to nitropyrenes consistent with such a reaction was observed {i.e. susceptibility to digestion by SE nuclease, decrease in the temperature thermal helix-to-coil transition profile; unpublished results). ‘These f~d~gs led to the suggestion that nitropyre~es are indeed conver~d by bacteria to the ~o~espondi~g hydroxylamines which, following este~i~atio~,

, /

I

revlnmole

0 0.001 0.003 0.01 0.03 0.1 0.3 I.0

revlnmole

0.001 0.003 0.01 0.03 0.1 0.3 31.0

0

6wP~te)

The roman numerals in parenthesis refer to the &uctural

---

..-

2

a 10 25 81 377 734 628 783 3300

37

20 100

6.0

3 4 5 6 19

4 3 3 4 14 30 62 109

TAX977

3 63 182, 611 1294

TA1537

1350

358 444 663

4 6 6 24 136

15 650

7 39 116 457 1284 1909

TA1538

3.0

5 7 5 8 12

4

7.1

5 4 8 9 25

4

TA1978

7700

4 19 75 191 589 1198 1444 1592

31400

22 111 323 954 1571 1715

TAYS

formulae.

0

11 7 9 12 9 9

8

97

169

27 25 22 20 28 54

WP2uvrA

used resting (ove~~t}

5200

6 21 62 129 442 99s 1471 1614

28 330

15 107 305 871 1685 1856

TA98NR

growing cultures (ca. 2 X 101 per ml). The assays indicated as TM%*

760

0

used exponentislly

93 82 97 102 167 289 372 609

5750

75 89 130 248 514 968

TAlOO

7 9 8 12 9 9 8 IO

0

13 9 7 10 14 14

TAl.535

FOR BACTERIA

Concentration

OF NITROPYRENES

AR assays ercept those marked TAS8*

w

1,3prrinitxopyr~ne

Compound

MUTAGENICITY

TABLE 1 (continued)

cultures.

16 590

21 51 108 379 983

40 700

12 344 403 1163 1600 X060

TAM *

192 y*2 I

act as electrophiies. It is hypothesized further that this biotransformation is carried out by nitroreductases other than the “classical” nitroreductases (i.e. the ones that recognize nitrofurans and have been characterized previously). Evidence in support of t,his hypothesis comes from the finding that 4-nitroquinoline-l-oxide and the nitroacridine Entozon, both of which require reductioh of their nitro function to express their mutagenicity (Tada and Tada, 1976; Nagao and Sugimura, 1976; Rosenkranz and Mermelstein, 1980; McCoy et al., 1981), exhibit full genetic activity in the tester strain deficient in ‘“classical” nitroreductace (Rosenkranz and Mermelstein, 1980; McCoy et al., 1981). Recently, we isolated a bacterial strain that appears to be deficiet:t in the nitroredu~t~e that reduces the mtro group of nitrated poly~y~li~ aromatic hydrocarbons including nitropyrenes. This strain, while fully responsive to nitrofurans, niridazole and 4-nitroquinoline-l-oxide, is not optimally mutagenized by 3 of the 6 nitropyrenes (McCoy et al., 1981). There are a number of features related to the mutagenic activity of the nitropyrenes which are unusual and require further elaboration. It should be noted that the mutagenic activities of 1-nitropyrene for some of the tester strains recorded in the present study (Table 1) are lower than those reported by us previously (Rosenkranz et al., 1980a, 1981). ‘It has been brought to our attention that the previously studied specimen contained a small amount of several other nitrated species. We have reason to believe that isomeric dinitropyrenes were the mutagenic impurit.ies. Because of the potent mutagenic activity of dinitropyrenes (Table 1), z total of 0.8-1.5% of these species would account, for the differences in mutagenic activities between the prior and the present results. We also note that 1-nitro-(I), 1,3-dinitro(I1:) and to a limited extent 1,3,6,8tetranitro-pyrene (VI) are recognized bly the “classical” nitroreductase, i.e. their

m~~tagenicity is reduced wLlen tested in strain TA~$~~~ while the other bers of the group are not, Ap~~e~tly only nitro ~~bs~t~t~~~ in ring A of the pyrene mule~ule (I, II) is re&o~ized by the %sssical” nitru~~d~~~se* abide nitro substiWions in rings A and C require the ~~tici~at~o~ of an a~t~~~~t~ nitroreductase for expression of mutagenicity. It remains to be esta cliched whether these dif~~~e~~es reflect sterie factors, redox potenMs or possibly other causes, but further studies are underway ~0 elucidate this phe~~rn~n~n. The obs~r~tion that the relative rnn~ge~i~ ~~~t~~~ies irt strain TA~~3~ do not parallel those found in TAQE: is novel as well. Strain TASS differs from TAlEi by the presence of a plasmid ~~~~~~1~ coding for an e~or-~r~n~ repair e~~yrne fGozs and Devuret, 3977; McCann et al., 19’95; ~~~ti-~~~~~d~~ et al,, lW7; Todd et al., 1979; Walker, X9%8). Generally this results in an increased mutagenic response. Finally, the observation tkat 1,8-dinitro~~~~~~ (IV) is much mor2 mutagenic fcr T-498 than the other nitropyrenes is e.lso nuzzling as a priori no struct~al feature seems La account for this ~~t~~t activity, Possibly, the DNA adduct formed by this isomer is recognized more readily by the error-prone DNA-repair enzyme, Also ~otewo~hy are the observations regarding the ~~~~~n~c~~y of the v*aious nitro~yre~es for the different tester strains, J[n strain TA1538, the bigbest values are obtained for 1,3~ini~o- (II) and 1,3,6-~nitro-pyrene (V), foll~~ved by 1,6dinit~o- fIII), 1~8di~it~o- (IV), ~~3,6,~-tetr~itrQ- (VI) and 1-nionovitro-pyrene /Ef, In ~~nt~~st~ in strain TA98 and ~A9~~~~ the successivrt addition of nitro groups results in increased activity which reaches a m~imum with 1,3,6_trinitropyrene (V) and then declines with J.~3,6,‘~-te~anitr~~yren~ [VI). Such a finding was Z.&Wmade with the nitr~~~~~~ne series (Xosenkranz ahd ~ermelst~in, 1980) and might reflect steric hinderanees in the enzymic reduction to hydroxylamine or the subsequent adduct form.ation with DNA due to the excessive number of nitru functions. It is noteworthy that ~,3~6-~initr~~yre~e (V) was the onfy member of the group that was mutagenic for E. cofi ~PZ~~~A, suggesting that under detail ~~curnstat~~es it might be endowed with base-substitution act Sty. Cer~inly the s~~tural basis of the mutagenic action of nitrop~~nes is worthy of further study. Most of the experiments recorded in Table 1 were obtained with exponentially growing bacteria. The use of growing bacteria had been indicated as the result of a large coll~b~~tive study in which it was found that greater intraas well as inter-laboratory reproducibility was obtained when such bactevia were used fCheli et al., 1980; Rosenkranz et al,, 2980b). In the present study of nitropy~enes~ huw~v~~~ it was found that the use of exponentially cowing bacteria resulted irn variable results when di-, tri- and tetra-pyrenes were tested while the use of ove~ight cultures not only yielded more reproducible results but also of enhanced specific acti~t~~s (Table 1, last column). We hypothesize that this reflects tbe state of anaerobiosis of the bacteria. overnight ~resti~~~ cultures having exhausted some of their nu~ients enter the ferm~ntativ~ state, The putative hydroxylamine intermediates formed from polynitropyrenes WE? presumably ~xyge~-~~b~l~ ,tassuch hy~oxylamin~$ are known to revert, ~xP enzymatically, to the oxidized nitro form. It is possible, therefore, that the

191

reduced oxygen tension of fermenting bacteria will favor the formation of the penultimate hydroxamates thus resulting in increased mutagenicity. 1,8-Dinitropyrene (IV) exhibits the highest mutagenicity recorded in the literature varying between 72 900 and 254 000 revertants per nanomole for growing and resting cells, respectively. This activity should be compared to that of Trp-P-2 (3-amino-l-methy!-5H-pyrido[4,3-blindale) in the presence of microsomal enzymes (2760 revertants per nanomole) heretofore considered the most rnu~gerll~ chemical reported in the literature {~ugimura and Nagao, 19’79). Nitro~onta~~g chemicals as a class are modestly potent bacterial mutagens and it has been suggested that this potency reflects the active nitroreductases present in enterobacteriaeceae (Ames, 1979; McCann and Ames, 1977; Rosenkranz and Mermelstein, 1980). As nitropyrenes are apparently transformed to biologically active intermediates by nitroreductases that are different from the “classical” ones, and that may bSe unique to bacteria (unpublished results), it might well be that their potent mutagenic activities in bacteria rellect the activity of these possibly unique bacterial enzymes. In view of the probable ubiquitous presence of nitrated polycyclic aromatic hydrocarbons in our environment (see Introduction), our studies to determine the structural basis of their potent mutagenicity and a determination of whether this activity reflects a purely microbial characteristic, may be of significance in assessing the possible risks posed by the presence of such substances in the environment. These studies are especially timely in view of the recognition of the presence of such nitrated poiycyclic aromatic hydrocarbons in diesel emissions. Acknotvledgements The authors are greatly indebted to Mr. Horace Becker, Xerox Corporation, for much encouragement and many stimulating conversations. They also wish to express their thanks to Drs. RN. Ames and M.H.L. Green for making available to them the Salmonella and E. co& teszer strains, respectively. References Ames, B.N. (1972) A bacteria1 system for detecting mutagens and carcinogens, in: H.E. Sutton and M.L Harris (Eds.j, Mutagenic Effects of Env~onment~ Contaminants, Academic Press, New York, pp. 5766. Ames. B.N. (1979) Identifying environmental chemicals causing mutations and cancer, Science, 204, 587-593. Amt-z, B.N., F.D. Lee and W.F. Durston (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sei. (U.S.A.), 70, ‘X2-786. Ames, RN.. J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Satmone~a/m~maf~n-microsome mutagenicity test, Mutation Res., 31.347-364. Cheli, C., D. DeFrancesco, L.A. Petrulio, E.C. McCoy and H.S. Rosenkranz (1980) SalmoneUa mutagenicity assay: Reproducibility, Mutatiou Res.. 74, 145-150. Fiich, W.L., and D.H. Smith (1979) Analvsis of adsorption properties and adsorbed species on eommercial polymers carbon, Environ. Sci. Technol.. 13, 341-346. Fitch, W.L., C.T. Everhart an4 D.H. Smith. (1978) Cha-acterization of carbon black adsorbates and a&. facts formed during extraction, Anal. Chem., 50.2122-2126. Gaze, A., and R. Devoret (1979) Repair promoted by plasmid pKMlB1 is different from SOS rep&, Mutation Res., 61. 163-179. Greet. 1M.H.L.. and W.J. Muriel (1976) Mutagen testing using trp* reversion in Eseherichio coli. Mutation Res., 38, 3-33.

Jager, J. (1978) Detection and characterization of nitro derivatives of some polycyclic aromat,ic by&ocarbons by fluorescence quenching after thin-layer chromatography: application to air poRution &n&ysis, J. Chromatog,, 152, 573-578. Jerina, D.M.. H. Yagi. R.E. Lehr, D.R. Thakker, M. Schaeffer-Ridder. J.M. Karle, W. Levm, A.W. Wood, R.L. Chang and A-H. Conney (1978) The hay-region theory of carcinogenesis by polycychc aromatic hydrocarbons,, in: H.V. Gelboin and P.Q.P. Ts’O (Eds.). Polycyclic Hyorocarbons and Cancer, Vol. I, Academic Press. New York, pp. 173-188. King, CM., C.Y. Wang and P.O. Warmer (1980) Evidence for the presence of nitro aromatics in air-born pazticulates. Proc. Am. Assoc. Cancer Res., 21. 83. Kubitscheck. H.E., and D.M. Williams (1980) Mutagenicity of fly ash from fluidbed-bed combuster during start-up and steady state operating conditions, Mutation Res., 77, 287-291. Ldfroth, G. 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Mermelstein (X981) Evidence for the existence of a family of bacterial. nitro reductases capable of activating nitrated polycyclics to mutagens, Environ. Mutagen., In press. Miller, J.A. (1970) Carcinogenesis by chemicals: An overview, Cancer Res.. 30, 559-576. Monti-Bragad~, C., S. Venturini and P.A. Todd (1977) Interaction between two error-prone DNA repair systems in Escf2erichia co& FEMS Mierobiol. Let&, 2, X25--128. Nagao, M., and T. Sugimura (1976) Molecular biology of the carcinogen 4-nitroquinoline-l-oxide, _4dv. Cancer Res., 23, 131-169. Pederson, TX., and J.-S. Siak (1980) Characterization of direct-acting mutagens in diesel exhaust particulates by thin-layer chromatography, Ann. Meet- Am. Chem. Sot. Abstr., Div. Environ, Chem. Pitts Jr., J.N. (1979) Photochemical and biolog:cal implications of the atmospheric reactions of amines and benzo[a]pyrene. Phil. Trans. R. Sot. (London), Ser. A, 290, 55X-576. Pitts Jr., J.N.. K.A. van Canwenberghe, D. Grosjean. J.P. Schmid, D.R. Fitz, W.L. Belser Jr., G.B. Knudson and P.M. Hynds (1978) Atmospheric reactions of polycyctic aromatic hydrocarbons: Facile formation of mutagenic nitro derivatives, Science. 202, 515-519. Rosenkranz. H.S.. and R. Mermelstein (1980) The Salmonella mutagenicit:! and the E. coli To1 A+/Pol .4 Irepair assays: Evaluation of relevance to carcinogenesis in: G.M. Williams. R. Kroes, H.W. Waaijers and K.W, van der Poll (Eds.), The Predictive Value of In Vitro Short-Term Screening Tests in the Evaluation of C~cinogenicity, Else~erfNorth-HoB~d, Amsterdam pp. 5-26. Rosenkranz, H.S., and L.A. Poirier (1979) An evaluation of the mutagen:city and DNA modifying activity in microbial systems of carcinogens and non-carcinogens, J. Natl. Cancer Inst.. 62.873-892. Rosenkranz, H.S., and W-T. Speck (1975) Mutagenicity of metronidazole: activation by mammalian liver microsomes, Biochem. Biophys. Res. Commun., 66, 520-525. Rosenkranz, H.S., EC. McCoy, D.R. Sanders, M. Butler, D.K. Kiriazides and R. Mermelsteir. t1980a) Nitropyrenes: isolation, identification and reduction of mutagenic imQurities in a carbon black and toners, Science, 209. 1039--1043. Rosenkranz, H.S., G. Karpinsky and E.C. McCoy (1980bf Microbial assays: Evaluation and aPPliWtiOn to the elucidetion of etiology of cancer, in: K. No~hpoth and R.C. Garner ,(Eds.), Short-Term Mutalenicity Test Systems for Carcinogens. Springer, Bedin, PP. 19-57. Rosenkranz. H.S., E.C. McCoy, R. Mermelstein and W.T. Speck (1981) A cautionary note on the use of nitroreductase-deficient strains of Salmonella typhimurium for the dete-tizrn of nitroarenes as mutagens in complex mixtures including diesel exhausts, Mutation Rt%., 91, 103-195. Schuetzfe, D.. T.J. Prater, T. RiIey, A. Durisin and I. Satmeen (1980) Analysis of nitrated derivatives of PAH and d,L:ermination of their contribution to Ames assay mutagenicity for dies ?I particulate extracts, Fii-1h Int. Symp. Polynuclear Aromatic Hydrocarbons, Columbus, Ohio, Abstr. 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196 Todd, P.A., C. Monti-Bragadin and B.W. Glickman (1979) MMS mutagenesis in strains of Escherichio coli carrying the R46 mutagenic enhancing plasmid: phenotypic analysis of Arg+ revertants, Mutation Res.. 62.227-231. Tokiwa. H.. R. Nakagawa. K. Morita and Kamachi (1979) Andysis of mutagenic nitro compounds in environmental samples (in Japanese). Environ. Mutagen Sot.. Jpn., Abstr., p. 18. Walker, G.C. (1978) Isolation and characterization of mutants of the plasmid pKMlO1 deficient in their ability to enhance mutagenesis and repair, J. Bacterial.. l33, 1203-1211. Wang, Y.Y.. S.M. Rappaport. R.F. Sawyer, R.E. Talcott and E.T. Wei (1978) Direct-acting mutagens in automobiie exhaust, Cancer Lett.. 5. 39-47. Wei. E.T.. Y.Y. Wang and S.M. Rappaport (1980) Diesel emissions and the Ames test: A commentary, J. Air Poll. Control Assoc.. 30, 267-271.