Mutagenicity and genotoxicity of nitroarenes

Mutation Research, 114 (1983) 2 1 7 - 2 6 7

217

Elsevier Biomedical Press

Mutagenicity and genotoxicity of nitroarenes All nitro-containing chemicals were not created equal Herbert S. Rosenkranz

~

and Robert Mermelstein

2

I Center for the Environmental Health Sciences and Department of Epidemiology and Community Health, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 (U.S.A.); and z Joseph C. Wilson Center for Technology, Xerox Corporation, Webster, New York 14580 (U.S.A.) (Received 25 M a y 1982) (Revision received 8 S e p t e m b e r 1982) (Accepted 10 S e p t e m b e r 1982)

Contents Summary .......................................................... I. Introduction .................................................... II. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. R e a c t i o n with D N A in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Preferential i n h i b i t i o n of D N A repair-deficient b a c t e r i a . . . . . . . . . . . . . . . . . . . . . V. Prophage induction ........................................... ~... VI. M u t a g e n i c i t y in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Reverse m u t a t i o n s in S a l m o n e l l a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. R e p r o d u c i b i l i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Purity of the test chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. M u t a g e n i c specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Effects of substituents on the m u t a g e n i c i t y of nitroarenes . . . . . . . . . . . . . . . . 5. Effects of the presence of p l a s m i d o n the m u t a g e n i c i t y of nitroarenes . . . . . . . B. F o r w a r d m u t a t i o n s in S a l m o n e l l a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Basis of the m u t a g e n i c i t y in b a c t e r i a of nitroarenes . . . . . . . . . . . . . . . . . . . . . . . . . VII. G e n e t i c activity in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. (3enotoxic and genetic effects of nitroarenes in cultured m a m m a l i a n cells . . . . . . . . . A. I n d u c t i o n of D N A repair synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Preferential i n h i b i t i o n of D N A repair-deficient cells . . . . . . . . . . . . . ........ C. I n h i b i t i o n of D N A synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '., ... D. Sister-chromatid exchanges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ E. C l a s t o g e n i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. M u t a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3. S o m e u n e x p e c t e d o b s e r v a t i o n s c o n c e r n i n g genetic effects of nitroarenes on cultured m a m m a l i a n cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Cell t r a n s f o r m a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. A b i l i t y of 2 - n i t r o f l u o r e n e to enhance m o u s e l e u k e m i a virus m u l t i p l i c a t i o n . . . . . . . IX. N a t u r e of the D N A a d d u c t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. M i c r o b i a l m e t a b o l i s m of nitroarenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . •. . . .

0 1 6 5 - 1 1 1 0 / 8 3 / 0 0 0 0 - 0 0 0 0 / $ 0 3 . 0 0 © 1983 Elsevier Science Publishers

218 218 219 221 223 223 223 223 223 225 226 234 235 235 237 242 243 243 243 243 243 244 244 246 246 247 248 249

218 xI.

Relationship between mutagenicityin bacteria and biotransformation by mammalian enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. Biotransformationof nitroarenes in animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Studies with whole animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Induction of sperm-head abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Host-mediatedassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIV. Carcinogenicityin animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XV. Somethoughts on nitroarenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... ,.

250 253 254 254 255 255 256 256

Summary The nitrated polycyclic aromatic hydrocarbons constitute a group of chemicals of environmental concern which display a broad spectrum of mutagenic, genotoxic and carcinogenic properties. Some members of the group are the most potent direct-acting bacterial mutagens while others exhibit low levels of potencies which require metabolic activation mixtures. Bacterial mutagenicity is dependent upon reduction of the nitro function. In mammalian cell systems the genetic and genotoxic effects of these nitrated chemicals include the induction of unscheduled D N A synthesis, sister-chromatid exchanges, chromosomal aberrations, gene mutations and cell transformation. The qualitative as well as quantitative expression of these effects is dependent upon the species and tissue of origin as well as culture history of the cell which in turn determine their enzymic capabilities and the conversion of these nitroarenes to ultimate mutagens and genotoxicants. In eukaryotic cells the following bioactivation pathways have been recognized: (a) reduction of the nitro moiety, (b) ring oxidation (the nature of which is influenced by the nitro function) followed by reduction of the nitro group, and (c) ring oxidation without concomitant reduction of the nitro moiety.

I. Introduction The nitrated polycyclic aromatic hydrocarbons, or nitroarenes, are a group of fused ring aromatic hydrocarbons (see structural formulae), which contain one or more nitro moieties covalently linked to cyclic carbon atoms. An examination of the rnutagenic and genotoxic properties of the nitroarenes appears to be timely in view of the recent recognition that this group of chemicals may well have a widespread distribution in the environment. Although the pharmacological and toxicological properties and the environmental persistence of monocyclic nitrated aromatic chemicals (e.g., chloramphenicol, nitrated benzenes and nitrated toluenes) and nitroheterocyclic nitrated aromatic chemicals (e.g., nitrofurans, nitroimidazoles) have been the subject of numerous studies, the nitroarenes, which are the subject of the present review, have only recently begun to be examined. The chief impetus for this interest

219

in the biological properties of these ,chemicals may well have been the findings of their facile formation in the environment (Pitts et al., 1979) and their unexpectedly potent mutagenicity for microorganisms (Rosenkranz et al., 1980b). The present review will be concerned with the mutagenicity and genotoxicity of nitroarenes and nitroazaarenes. Only fused ring structures will be considered, the nitrated benzenes, toluenes and biphenyls being excluded as are the nitroazaarene-N-oxides such as 4-nitroquinoline 1-oxide and its congeners.

II. Occurrence

Present indications are that nitroarenes are a by-pr0duct of incomplete combustion processes, thus, with the exception of acts of nature, such as forest fires and volcanic eruptions, their presence in the enviroment appears to result from man-made activities. Nitroarenes have been detected in the extracts of diesel and gasoline emissions, fly ash particles, carbon blacks, cigarette smoke condensates, and emissions originating from incinerators, residential home heaters and wood burning stoves and in the urban atmosphere (Agurell and Lrfroth, 1982; Campbell et al., 1981; Clark et al., 1982; Erickson et al., 1981; Fukino et al., 1982; Gibson, 1981; Henderson et al., 1981; Lewtas et al., 1981, 1982b, Li et al., 1982; Ltffroth, 1981, 1982; McCoy and Rosenkranz, 1982; Mumford and Lewtas, 1982; National Academy of Sciences, 1981; Nishioka et al., 1981; Pederson and Siak, 1981a, b; Peterson et al., 1981a, c; Pitts et al., 1982a, b; Rappaport et al., 1980; Riley et al., 1981; Salmeen et al., 1982; Schuetzle et al., 1981, 1982a, b; Talcott and Harger, 1981; Wei et al., 1982; Xu et al., 1981a, b, 1982; Yergey et al., 1981; Zweidinger, 1981). Because of the anticipated shift to the use of improved fuel efficient diesel engines in passenger vehicles with an attendant increase in particulate emissions (Ingalls and Bradow, 1981; U.S. Environmental Protection Agency, 1978), it is not surprising that diesel emissions have been the subject of numerous recent studies. In excess of 60 nitroarenes have been detected in diesel emissions (Henderson et al., 1981; Riley et al., 1981; Xu et al., 1981). In this class of chemicals, 1-nitropyrene appears to be the most abundant, while the dinitropyrenes may be the most potent bacterial mutagens (Pederson and Siak, 1981a, c; Pitts et al., 1982b; Salmeen et al., 1982; Petersen et al., 1981; Henderson et al., 1981; Campbell et al., 1981). Nitroarenes are readily formed when products of incomplete combustion (polycyclic aromatic hydrocarbons), oxides of nitrogen and traces of acid are present simultaneously (Lrfroth et at., 1981; Pitts, 1979; Pitts et al., 1979; Tokiwa et al., 1981a, b). Present indications are that the nitrogen atom of nitroarenes originates from atmospheric nitrogen present during or after combustion. Thus operation of a diesel engine in the absence of nitrogen (in an atmosphere of argon) does not result in the formation of nitroarenes (Yergey et al., 1981). Moreover the nature and quantity of nitroarenes produced in the combustion process is independent of the fuel used. Thus operation of a diesel engine with either pure aliphatic hydrocarbons or with No. 2 diesel fuel yields the same distribution of nitroarenes (Yergey et al., 1981). The mutagenic activity of diesel emissions appears to be a function of the temperature of the combustion process as well as of the

220 8

@ 9

I

5

~0

I

8

4

5

NAPHTHALENE ANTHRACENE

9 4

I

2

I

~

6

5

FLUORENE ACENAPHTHENE

2 9 I0 I

~ z

9 I0

8 ~ 5

12

I 2

B

~ 9 8

7 6

P~NAN~RENE FLUORA~ENE

t

5

~

5

7

PYRENE

6

CHRYSENE 2

I~3

,,

8

7

6

7

6

5

s~1's 7

BENZO(e)PYRENE

EENZ(o)ANTHRACENE BENZO(o)PYFENE 2 I 1

I 0

II 12

~ ~ 5

l

2

12

9 8

4

9

4

6

8

PERYLENE

I

'of~y~y~)3

4

7

BENZO(g,h,i)PETh'LENE

6

~ENE CH3 - CH--(CH2)3- ~C2H5)2" 2HCi NH I

CI~

OCH3

I CH2-CH(OH)-CH2- N(C21..15)2• 2HCI

I

NH N

OCH3

l---'-t

N 0 2 ~ S ~ - - N',IfN~H 0

-m-

~N02

I

3Z

0

v'r /CH?.-CH2

/CH3 CH2-CHz-N_ I ~CH3 O~NO

vrr

3ZE

2

221 configuration of the engine. In view of the numerous and various types of stationary and mobile emission sources, it is not surprising that nitroarenes have been detected n o t only in heavily travelled confined areas such as tunnels but also in urban environments, such as the cities of Prague, N e w Y o r k City, Los Angeles and Philadelphia (J~iger, 1978; Jungers et al., 1981; Lewtas et al., 1981, 1982b; Pitts et al., 1982a).

IlL Reaction with D N A in vitro There is no evidence that exposure of purified D N A to nitroarenes results in covalent attachments to the deoxypolynucleotide moiety. Thus, exposure of nitroarenes (nitronaphthalenes, nitrofluorenes and nitropyrenes) to D N A did not lead to decreases in the thermal helix-to-coil transition profiles (T m) ( M c C o y et al., 1981b, d; Mermelstein et al., 1981; Rosenkranz et al., 1982b). N o r did exposure to nitropyrenes result in spectral shifts, an effect on the sedimentation behavior of closed circular D N A , or in an increased sensitivity to digestion with S 1 nuclease (Mermelstein et al., 1981). Finally, treatment of D N A with 2-nitrofluorene or a mutagenic extract from diesel emissions containing nitroarenes did not result in a sequestration by the D N A of the mutagenic activity (Pederson and Siak, 1981a). There is, however, an indication that some nitroarenes and nitroazoarenes are capable of intercalating between D N A based pairs as evidenced by increases in the

TABLE 1 EFFECT OF NITROARENES ON THE THERMAL HELIX-TO-COIL TRANSITION OF DNA Chemical

DNA nucleotide/ ligand

Tm (°C)

+ ATm

Reference

McCoy et al., 1981b McCoy et al., 1981b McCoy et al., 1981b McCoy et al., 1981b McCoy et al., 1981b

None 2-Nitronaphthalene 3-Nitro-l,8-naphthalic anhydride (VI) M4212 (VII) M12210 (VIII) 1,3,6,8-Tetranitronaphthalene

100 100 100 100 100

69.9 2.5 0.8 7.3 12.2 2.2

2-Nitrofluorene 2,7-Dinitrofluorene 2,7-Dinitro-9-fluorenone 2,4,7-Trinitro-9-fluorenone 2,4,5,7-Tetranitro-9-fluorenone

100 100 100 100 100

0.2 2.1 3.2 4.4 1.8

Entozon (III) 9-Aminoacridine (I)

80 400

16.5 6.8

i-Nitropyrene 1,8-Dinitropyrene 1,3,6-Trinitropyrene

100 100 100

0 0 0

McCoy et McCoy et McCoy et McCoy et McCoy et

al., 1981d al., 1981d al., 1981d al., 1981d al., 1981d

McCoy et al., 1981c McCoy et al., 1981c Rosenkranz et al., 1982b Rosenkranz et al., 1982b Rosenkranz et al., 1982b

222

T m and by spectral shifts. Thus, nitroacridine derivatives [e.g., Entozon (III)] exhibit such propeorties (Table 1; Kalinowska and Chorazy, 1980; McCoy et al., 1981c). This is not surprising in view of the structural analogy of these chemicals with known DNA intercalators such as quinacrine (II) and 9-aminoacridine (I). However, other nitroarenes unexpectedly also exhibited this property to a limited degree. Thus, nitronaphthalenes, nitrofluorenes and specifically 2,4,7-trinitrofluorenone (Table 1) cause increases in the T m. In the case of the nitrofluorenones, this may well involve a cooperative effect of the carbonyl function at position 9 as exemplified by the fact that 2,7-dinitro-9-fluorenone caused a greater increase in the T m than did 2,7-dinitrofluorene (Table 1). Introduction of an anhydride or a substituted imide moiety onto the nitronaphthalene molecule (VI, VII and VIII) also facilitates intercalation as evidenced by thelncrease in T m (Table 1 and BrSna et al., 1980), by spectral shifts and by reversal of supercoiling of covalently closed circular DNA (Waring et al., 1979). It might be noted (and will be discussed later), that: (1) Even though the majority of nitroarenes do not form covalent linkages with isolated D N A and although they are direct-acting mutagens for Salmonella (i.e., they do not require exogenous activation by microsomal enzymes), nevertheless they are metabolized by the bacteria to intermediates capable of forming adducts with the cellular DNA. (2) Although some of the nitroarenes appear to be capable of intercalating between D N A based pairs (see above) and even though nitroarenes induce mainly mutations of the frameshift type (see below), this activity is not primarily due to intercalations between DNA based pairs but actually to DNA adduct formation.As will be pointed out below, the frameshift activity that these agents induce as a result TABLE 2 EFFECT OF N I T R O A R E N E S ON T H E G R O W T H OF DNA R E P A I R - D E F I C I E N T BACTERIA Chemical

Bacteria

DNA repair system

Results

Reference

2-Nitronaphthalene

E. coli

PolA +/PolAI

+

2-Nitrofluorene

E. coli

PolA +/PolAi

+

E. coli

uvrA + recA +/uvrArecA uvrA +/uvrA uvrA + polA +/uvrApo IAI uvrA + lexA +/uvrAlexA recA +/recA 1 recA +/recA 13 recB + recC +/recB21 recC22 recA + recB + recC + / recA 13recB21 recC22 rec +/recA45 rec +/recA45 hcr + recA +/hcr42recA 1

+

Rosenkranz and Poirier, 1979 Rosenkranz and Poirier, 1979 McCarroll et al., 1981b McCarroll et al., 1981b McCarroll et al., 1981b McCarroll et al., 1981b Suter and Jaeger, 1982 Suter and Jaeger, 1982 Suter and Jaeger, 1982 Surer and Jaeger, 1982

B. subtilis

-

-

+ + +

McCarroll et al., 1981a Suter and Jaeger, 1982 Suter and Jaeger, 1982

223 of intercalations is rather low and can be calculated from the activities exhibited in strains TA1537 and TA1977.

IV. Preferential inhibition of DNA repair-deficient bacteria Only 2-nitronaphthalene and 2-nitrofluorene have been tested for their ability to preferentially inhibit the growth of DNA repair-deficient microorganisms. Both of these chemicals appear to act preferentially on DNA repair-deficient E. coli and Bacillus subtilis (Table 2). However, it should be noted that this activity is dependent upon the type of lesion in the DNA repair pathway. Because these DNA repair-deficient strains are inhibited preferentially by chemicals that are DNA-damaging (Rosenkranz and Leifer, 1980), the conclusion can be drawn that these two nitroarenes are genotoxic.

V. Prophage induction Prophage induction is thought to be a measure of DNA damage followed by the induction of an error-prone DNA repair process (SOS pathway). As such, it has been suggested that prophage induction might be a useful prescreen for the detection of potential carcinogens (Moreau and Devoret, 1977). Modelled after the tester strains developed by Ames and his associates (1975), E. coli tester strains with deficiencies in envelope lipopolysaccharide have been constructed (Moreau et al., 1976) and these exhibit increased sensitivity to the prophage-inducing capacity of larger molecules. Using such an envelope-defective E. coli strain, 2-nitrofluorene was reported to induce prophage ~ (Ho and Ho, 1981). The nitroazaarene Ledakrin [1-nitro-9-(3'-dimethylaminopropylamino)acridine dihydrochloride] was also found to be a prophage inducer (Gajcy et al., 1979).

VI. Mutagenicity in bacteria A. Reverse mutations in Salmonella 1. Reproducibility Although the Salmonella mutagenicity assay has been well described (Ames et al., 1975) and even though the procedures have been carefully standardized (De Serres and Shelby, 1979), it was found that nitroarenes exhibited a greater variability in their responses upon repeated testing under presumably identical conditions (Rosenkranz et al., 1980a, 1982c; Cheli et al., 1980) than other compounds. It should be noted, however, that the variability is mainly qualitative, i.e., one of mutagenic potency. Only two of the chemicals listed, 1,8-dinitronaphthalene and 6nitrobenzo[a]pyrene, are reported as mutagenic by one group of investigators and to be inactive by another. In a multi-laboratory collaborative study, the Salmonella

224

mutagenicity assay was found to yield the most reproducible results when using growing cultures (Dunkel and Simmon, 1980). However, application of this test procedure to nitroarenes even in an intralaboratory comparison resulted in a widely variable response. Thus a distribution of mutagenic signals in response to a single dose of 2-nitrofluorene (100/~g per plate, each dose done in triplicate) revealed an unusually broad distribution (Fig. 1, see also Anders et al., 1982). This heterogeneity in response together with the uncertainty inherent in single dose determinations, even when done in triplicate, led to determinations of the mutagenic potency of 2-nitrofluorene from the linear (ascending) portions of the dose-response curves. When such analyses are carried out for most chemicals, satisfactory reproducible results that can be used for structure-activity relationships are obtained. For 2-nitrofluorene, however, the experimental variability still remained (Fig. 2). The results of a further set of 18 dose-response curves obtained with the same chemical substantiated this variability (Table 3). A careful analysis of the results revealed (Rosenkranz et al., 1982c) that this was due to the fact that the indicator microorganisms were at different stages of growth. While this had no effect on non-nitrated chemicals, it greatly affected the results obtained with nitroarenes. In a systematic study of this phenomenon, it was found further that the most reproducible results with nitroarenes were obtained when resting cultures were used. In the case of nitropyrenes this was accompanied by up to a 4-fold increase in the specific mutagenicity of these chemicals (Mermelstein et al., 1981). (This increase in mutagenicity appears to be due to a stabilization of the penultimate mutagen, the arylhydroxylamine, see below). IZ.$i

!

i ..... !

.! T

15,75

T

?,S!

iE F

6.2S

+

$.le

*

g 17 N ¢ Y

i lamM

3.7~i • I

I m l l

• •

Ju w w u n•n•lw

mM H•

• n •

2.SO •

• n H n H n•uu n unmHn un•~u miNmm ~mn~mmNm mlmm~l ~ m • • • • ii uu ~ • m m n u m m Nmmmwn m • • •• • • m n l V m H W U N m m • m n ~n • 1.15 I u IIIIlIIRI uluulumllllllllllmn iii I umw•nunwmnuuuuauu~mulnumunuuun • • w uulmulaii miami nnumiaunnniilaufllnuum an • • ag • • a n • n m•m u• w n I I I N u I w N I I u l I I I U I U H I N I I a I u u gmmm i Ill INi lliRUlilliUmUmmnNmlmNmmNnmmmlmmu muuumummmu • Im | ~ - - m l l - l m - $ m u l - m u l u m m u a u m m u a a l u u u u m N N a a u a n N m m u u a N u N i m • u m u m m u u m u l l . m ~ - ,.-.,.~. . . . I

IlI.IO

qIl.$I

$II.II

II|.IO

$OOI,II 121I,IO REVI~T&NTSJPLAT!

14l$.II

• m-$-I ....... IlO$.lO

$ ........ IlOt.OI

ZO$|.OI

Fig. 1. Response of Salmonella typhimurium TA98 to 2-nitrofluorene (100/xg per plate, done in triplicate). N = 8 9 6 ; m e a n = 6 7 4 . 7 + 8.2.

225

2-NITROFLUORENE; TA98

I000

• 1o-z7-77

•s-1-re

',ll-7-'r7

.S-2-Ta

x 3 - 8 -78

v 8-23-78/

• z-,,-r8

/

• 7-28-~,8 /

/

/ /

Z"

,,,~

/..

=. fl::®

500

i

i

I

IO p.g per plot==

I

IOO

Fig. 2. Mutagenicity of 2-nitrofluorene for Salmonella typhimurium TA98; dose-response relationships.

2. Purity of the test chemials Another possible cause of error may relate to the purity of the test chemicals. In most instances, nitroarenes are obtained by nitration of the parent polycyclic aromatic hydrocarbon. Frequently the starting hydrocarbon contains a number of impurities such as homologs or isomers. Because this procedure results in a mixture of chemicals differing in extent of nitration (Rosenkranz et al., 1980b; Campbell et al., 1981) this then requires fractionation of the products. While both gas liquid chromatography and high pressure liquid chromatography procedures have been used, the latter technique appears to be the method of choice. In most instances, it was found that dinitroarenes are much more mutagenic than their mononitro analogs (e.g., nitronaphthalenes, nitrofluorenes, nitropyrenes) (Table 4), which implies that the separation of the mononitroarenes from the polynitrated species must be rigorous. This is not always the case, especially when using samples of commercial origin. Thus, in a recent study, 1-nitropyrene was reported as exhibiting a mutagenicity in strain TA98 of 2000 rev/nmoles (Rosenkranz et al., 1980b). Subsequently, it was found that this preparation contained approximately 1% dinitropyrenes which accounted for about 80% of the activity; upon further purification to remove these trace impurities, 1-nitropyrene was found to exhibit an activity of 400 rev/nmole as well as a different response to a panel of nitroreductase-deficient microorganisms (Mermelstein et ai., 1981; McCoy et al., 1981e). Similarly it was found that 85% of the mutagenicity of a commercial sample of 3-nitro-9-fluorenone was due to dinitrofiuorene derivatives present therein (Jin et al., 1982). In view of the above-mentioned variability in specific activities and the differences in the purity of the test chemicals and various environmental mixtures, it is difficult to compare values obtained in different laboratories in attempting to draw conclusions related to the structural basis of the activities of these chemicals.

226

TABLE 3 M U T A G E N I C I T Y OF 2 - N I T R O F L U O R E N E FOR Salmonella typhimurium TA98 Expt.

Date

Rev./p.g

Expt.

Date

Rev.//.t g

1 2 3

3-13-78 7-11-78 9-08-78

79.3 60.1 45.7

10 11 12

9-25-79 10-29-79 2-26-80

61.6 85.7 43.4

4 5 6

10-19-78 4-02-79 4-12-79

79.2 65.6 26.7

13 14 15

2-26-80 4-08-80 5-16-80

47.2 44.6 49.7

7 8 9

5-22-79 6-18-79 8-13-79

34.4 45.7 43.8

16 17 18

5-26-80 8-19-80 ! 1-17-80

22.9 40.1 24.8

Mean 50.0 + 18.6. DMSO control 11.9 + 6.6.

3. Mutagenic specificities The mutagenicity of nitroarenes for bacteria has been studied in greatest detail in the Salmonella mutagenicity assay developed by Ames and his associates (1975), although studies in the E. coil WP2uvrA system (Green and Muriel, 1976) have been reported as well. The data of the mutagenicity of nitroarenes reported in the literature are summarized in Table 4. [In addition to the purified chemicals listed in Table 4, the nitration products (i.e., mixtures) of anthracene, fluoranthrene, pyrene, perylene, chrysene, phenanthrene, benzo[a]pyrene, benzo[e]pyrene, benz[a]anthracene, benzo[g,h,i]perylene and benzo[k]fluoranthene have also been shown to be mutagenic for Salmonella tester strains TA98 and TA100 (Campbell eta., 1981; Rosenkranz et al., 1981).] A number of observations are immediately obvious: I. Bicyclic chemicals induced mainly mutations of the base substitution variety (TA1535) (e.g., nitronaphthalenes and nitroquinolines) while nitroarenes possessing three or more fused rings induce primarily mutations of the frameshift type (TA1538, TA98). This is substantiated by the responses of these chemicals in E. coil WP2uvrA which also responds primarily to mutagens which induce base substitutions (Brusick et al., 1980). II. There appears to be an optimal molecular size and configuration for maximal mutagenicity. Activity increases from the bicyclic to the tetracyclic ring system, thus, tetracyclic nitroarenes (nitrofluoranthenes and nitropyrenes) are the most mutagenic mononitroarenes reported. Pentacyclic nitroarenes show an abrupt decrease in direct-acting mutagenicity. Indicative of the importance of configurational effects, 2-nitroanthracene is about 7 times more active than 2-nitrophenanthrene in strain TA98. III. An examination of the mononitroarenes reveals a number of unexpected positional effects. Some of these are described below: (a) 2-Nitronaphthalene is more mutagenic than the isomeric 1-nitronaphthalene

227 and, moreover, while the 2-nitro isomer is mutagenic for tester strain TA1535, 1-nitronaphthalene requires the presence of plasmid pKM101 (e,g., strain TA100) to express its mutagenicity. (b) The same phenomenon is demonstrated in the nitroquinoline series. Thus, while 6-nitroquinoline is mutagenic in strain TA1535, 5-nitroquinoline requires the presence of the plasmid to express its mutagenicity. On the other hand, 3nitroquinoline is completely devoid of direct-acting mutagenicity. (c) In the carbazole series, dramatic positional effects were apparent as exemplified by the complete lack of activity of the 1-nitro isomer and the relatively potent activity of the 2-nitrocarbazole. (d) Studies in the same laboratory (i.e., minimizing experimental variability, Nilsson et al., 1981) show that 8-nitrofluoranthene is twice as mutagenic as 3-nitrofluoranthene in strain TA98, while in strain TA100 this is reversed: the 3-nitro isomer exhibits 7.5 times the activity of 3-nitrofluoranthene (Table 4). (e) In the same laboratory, 2-nitropyrene in strain TA98 exhibited almost 3 times the activity of the isomeric 1-nitropyrene. (f) While Pitts et al. (1982b) find that 6-nitrobenzo[a]pyrene is devoid of mutagenicity, they report appreciable direct activity for the isometric 1- and 3nitrobenzo[a ]pyrenes. (g) Similarly, there are significant differences in the mutagenic activities of 1- and 3-nitrobenzo(e)pyrene as well as between 4-nitro- and 7-nitrobenz[g,h,i]perylene (Table 4). IV. It is seen that an increase in the extent of nitration is paralleled by an increase in mutagenicity (for example, dinitronaphthalenes, polynitrofluorenes, and polynitropyrenes). However, tetranitro-arenes exhibit greatly reduced activities. Whether this reduction in activity is due to an inability to convert the tetranitroarenes to the corresponding active intermediates (presumably the arylhydroxylamines) or whether it reflects steric hindrance in forming DNA adducts following activation by the bacterial nitroreductases or the occurrence of another reaction, remains to be elucidated. V. The positional effects that were apparent with the mononitroarenes are even more dramatic when the dinitroarenes are examined. Thus, 1,8-dinitronaphthalene is devoid of mutagenicity while 1,5-dinitronaphthalene exhibits appreciable mutagenicity in strain TA100. Similarly, in the dinitropyrene series, dramatic differences among the various isomers are seen in the activities in strain TA98; moreover, the order of mutagenicities seen with that strain is different from that of its parent strain TA1538, which lacks the plasmid pKM101. Furthermore, the order of reactivities is still different in strain TA1537. VI. Some of the nitroarenes such as 1,8-dinitropyrene are among the most potent microbial mutagens reported in the literature. Only two other chemicals appear to approach its potency, one of these is a nitro-containing furan [2-nitro-7methoxynaphtho(2,1-b)furan (Weill-Thevenet et al., 1981)] while the other is a pyrolysis product present in broiled sardines which requires activation by microsomal enzymes to express its mutagenicity (Kasai et al., 1980; Nagao et al., 1981). VII. Although nitroarenes containing three or more rings induce primarily muta-

228 TABLE 4 N I T R O A R E N E S : M U T A G E N I C I T Y IN BACTERIA ( M U T A N T S PER N A N O M O L E ) WP2uvrA

TA1535

I -Nitronaphthalene

0 0

TA100 9.9 3.5 0.9 0.8 1.0

T A 1 0 0 + S9 1.0 0.5

2-Nitronaphthalene 4.3 1.1 .

.

.

3.7 - 0.30 1.4

.

1.2

4.8 - 1.16 1.3

2-Methyl- 1-Nitronaphthalene

0

1-Methyl-2-Nitronaphthalene

1.2

1.8

3-Methyl-2-Nitronaphthalene

0.2 7.2

0.2

14.1 4.7 13.2 0

8.3

0.2

1,3-Dinitronaphthalene 1,5-Dinitronaphthalene

0 1,8-Dinitronaphthalene 0 2,4-Dinitro- 1-naphthol

0

1,3,6,8-Tetranitronaphthalene

3-Nitro-1,8-naphthalic anhydride (VI)

6.8

0.4 0.3

0.03

5-Nitroacenaphthene 0.02 M4212 (VII) M12210 (VIII)

0.1

39.8 10.7 1.9

15.7 20.1 18.7

18

14

0.4 0.4

2-Nitrofluorene

0

8 - 4.7

0.03 3-Nitro-9-fluorenone

1.4

0.001

6 24

229

TA1538

-

TA98

TA98 + S9 1.1 0.4 0.2 0.2 0.05

0.1

1.0 0.8 0.9 0.4 0.4 0.2 0.87 0.2

0.4 - 0.04

0 0.2 1.0

0.3 0.2 0

6.1 2.9 2.5

0.8

-

65 9.9 10.5 7 34

18 72 31 32 88 47 51 2.8 4.9 70 14 41 383 47

0

Tokiwa McCoy Tokiwa McCoy

0.1 3.4 0.4

McCoy et al., 1981b McCoy et al., 1981b McCoy et al., 1981b

0.3

Tokiwa et al., 1981e Yahagi et al., 1975 Rosenkranz et al., 1982c

2.1 3.4

McCoy et al., 1981c McCoy et al., 1981c

0.2 7.5

18 12

-

0.8

E1-Bayoumy et al., 1981 E1-Bayoumy et al., 1981 E1-Bayoumy et al., 1981 McCoy et al., 1981b

2.2

17.3 10.5 6.8

14

72

0.04 31

Tokiwa et ai., 1981 Nilsson et al., 1981 E1-Bayoumy et al., 1981 Scribner et al., 1979 McCoy et al., 1981b

- 0.15 0.02 0 0.2 0.8

0.7 0.2 2.6

Reference

W a n g et al., 1980 Wang et al., 1978 Nilsson et al., 1981 EI-Bayoumy et al., 1981 Ho et al., 1981 Scribner et al., 1979 Simmon, 1979a McCoy et ai., 1981b

0.4

0.9 3.2 3.3 7.9 0

TA1537

et et et et

al., al., al., al.,

1981c 1981b 1981c 1981b

Tokiwa ** W a n g et al., 1980 W a n g et al., 1978 Bartsch et al., 1980 Pitts et al., 1982b Pederson and Siak, 1981a Scribner et al., 1979 Simmon, 1979a N e s t m a n n et al., 1980 Weill-Thevenet et al., 1981 McCoy et al., 1981d LaVoie et al., 1981c Pederson and Siak, 1981a Jin et al., 1982

230 TABLE 4 (continued) WP2uvrA

TA1535

TA100

2,7-Dinitrofluorene

6

0

0

2,7-Dinitro-9-fluorenone

2,4,7-Trinitro-9-fluorenone 2,4,5,7-Tetranitro-9-fluorenone 2-Nitroanthracene

T A I 0 0 + $9 14

180 6 530

0 0.03 0.03

0

457

2

220 t 59

5 794

287 1133

9-Nitroanthracene

2-Nitrophenanthrene 9-Nitrophenanthrene

0

1-Nitrofluoranthene

3.6

3.0

0.9

0.9

62 < 0.9

1.3

124

3-Nitrofluoranthene

7-Nitrofluoranthene 8-Nitrofluoranthene

25

1400 2 967

< 25

989 396

l-Nitropyrene

0

0

2-Nitropyrene

31

362

35

148

49

59 742

1,3-Dinitropyrene 0 1,6-Dinitropyrene

0

'~ 0

12159

52

55 420

120

0

1,8-Dinitropyrene

0

42 280

0

231 TABLE 4 (continued) TA1538

2931 346

TA98

TA98 + $ 9

5 180 2 560 920 471

TA1537

38 289 290

Tokiwa et al., 1981c Pederson and Siak, 1981a Levin et al., 1979 McCoy et al., 1981d

1 702

2160 1 227 1459

195

Levin et al., 1979 Jin et al., 1982 McCoy et al., 1981d

8 068 2 860

3 120 2125

214 572

Levin et al., 1979 McCoy et al., 1981d

2190 839

860 892

186

McCoy et al., 1981d Scribner et al., 1979

5 650

2.7 0.01 0.5 0.01 0.13

77

Reference

2.0

Tokiwa et al., 1981c Wang et al., 1978 Nilsson et al., 1981 Pitts et al., 1982b Pederson and Siak, 1981a Ho et al., 1981

0.5 0.01 0.22

145

128 < 0.5

Scribner et al., 1979 Nilsson et al., 1981

< 0.5

Nilsson et al., 1981

74 1286

13400 5 439 1434

9889

I 1 125

60 321

544 470 2 606 470 865 252

< 124

- 74

1508 54

80 *

67

54057 163 800 144760 126000 67 220

61 100 *

183 570

34700 *

265 966 195 774 274888 254000

Wang et al., 1980 Nilsson et al., 1981 Pitts et al., 1982b Pederson and Siak, 1981a Ho et al., 1981 Nakayasu et al., 1982 Mermelstein et al., 1981 Nilsson et al., 1981

2 225

78 960 *

Nilsson et al., 1981 Nilsson et al., 1981 Tokiwa **

57

499 453

Tokiwa et al., 1981c Nilsson et al., 1981 Pederson and Siak, 1981a

13400

Peclerson and Siak, 1981a Nakayasu et al., 1982 Mermelstein et al., 1981

33000

Tokiwa ** ~. . . . Nakayasu et al., 1982 Mermelstein et al., 1981'~

11800

Tokiwa ** Pederson and Sial~, 1981a Nakayasu et al., 1982 Mermelstein et al., 1981

70

241

232 TABLE 4 (continued) WP2uvrA

TA1535

TAI00

TA100+S9

1,3,6-Trinitropyrene 97

0

7 450 *

0

0

1 520 *

1,3,6,8-Tetranitropyrene

2-Nitrochrysene 5-Nitrochrysene

< 1.4 < 2.7

6-Nitrochrysene

97 273

96 5.5 192 4.0

7-Nitrobenz[ a ]anthracene

< 0.6

6-Nitrobenzo[ a ]pyrene

5 < 3

39 327

51

10.9

1-(or 3-)Nitrobenzo[ a ]pyrene 3-(or 1-)Nitrobenzo{ a ]pyrene l - N i t r o b e n z o [ e ]pyrene

<15

45

3-Nitrobenzo[ e ]pyrene

<15

< 30

208

< 60

- 892

3-Nitroperylene 4-Nitrobenzo[ g, h, i]perylene 7-Nitrobenzo[g, h,i]perylene I -Nitrocoronene 5-Nitroquinoline

<1 < 0.3 < 1.4 0

6-Nitroquinoline 0.18 8-Nitroquinoline 0 0 Entozon (III) 1-Nitro-9-(3'-dimethylaminopropylamino)acridine dihydrochloride

2-Nitro-9-(3'-dimethylaminopropylamino)acridine dihydrochloride

0.20 - 0.21 - 0.25 - 0.09 0.09 0 0 0

0

24

44

79 365

0

108400

0.2

49 < 0.6 3.5

- 0.5

2

9-(3'-Dimethylaminopropylamino) acridine dihydrochloride 1-Nitrocarbazole 2-Nitrocarbazole

0 0.5

0 O.l

233 TABLE 4 (continued) TA 1538

TA98

TA98 + $9

20300 *

238 547 40700

20100

Nakayasu et al., 1982 Mermelstein et al., 1981

2730 *

84551 15 590

3 300

Nakayasu et al., 1982 Mermelstein et al., 1981

< 0.6 < 0.6 269 12 < 14 0.3 31 < 1.5 61 0 1 567 1070 < 6

TA 1537

Nilsson et al., 1981 Nilsson et al., 1981

27 < 2.7

Tokiwa et al., 1981c Pederson and Siak, 1981a Nilsson et al., 1981

22 - 109

Nilsson et al., 1981

1.4

Tokiwa et al., 1981c Niisson et al., 1981 Wang et al., 1978 Pitts et al., 1982b

141 - 208 446

Pitts et al., 1982b Pitts et al., 1982b

1011 1 100

39

25

51

Nilsson et al., 1981

890

743

< 30

Nilsson et al., 1981

< 30

- 1784 282

Nilsson et al., 1981 Ho et al., 1981

- 1925 < 0.6 7.0

Niisson et al., 1981 Nilsson et al., 1981 Nilsson et aL, 1981

< 0.6 < 0.3 2.8 0.11 <0.14

Karpinsky et al., 1982 Chiu et al., 1978

< 0.04 0.14 0.05

Nagao et al., 1977 Chiu et al., 1978 Karpinsky et al., 1982

0

Nagao et al., 1977 Epler et al., 1977 Karpinsky et al., 1982

~ 0.08

0 288

Reference

338

10

43650

10912

37 200

132 000

108 800

Kalinowska and Chorazy, 1980

4

12

2

Kalinowska and Chorazy, 1980

0

0

0.2

Kalinowska and Chorazy, 1980

0 10.3

0 4.7

McCoy et al., 1981c Gajcy et al., 1979

LaVoie et al., 1981c LaVoie et al., 1981c

234 TABLE 4 (continued) WP2uvrA 3-Nitrocarbazole 4-Nitrocarbazole

TA1535

TAI00 0.1 0.5

TA100+ $9 0.4 0.8

* The values were normalized for resting cultures; all other data were for resting cultures. ** These values differ from the values originally reported (Tokiwa et al., 1981c) presumably because of the presence of impurities in the original specimens(H. Tokiwa, personal comm., 1982).

tions of the frameshift variety, i.e., they revert strains TA98 and TA1538, the specificity of this mutagenic response is different from that associated with the activity of agents that cause frameshift mutations as a result of intercalations between D N A base pairs. Thus, proflavin, 9-aminoacridine and 1,9-diaminoacridine, in the absence of light or of metabolic activation mixtures, induce mutations that are restricted to strain TA1537 (Table 5). There is no 'spillover' into strain TA98 or TA1538. On the other hand, chemicals which induce frameshift mutations not as a result of simple intercalations but as a consequence of the formation of covalent adducts with D N A (e.g., N-hydroxy-2-acetylaminofluorene, 7-bromomethyl-12methylbenz[a]anthracene, or 4-hydroxylaminoquinoline 1-oxide) without intercalating between D N A base pairs act primarily on strain TA1538 and its plasmid-containing derivative TA98. Such chemicals, however, also exhibit a 'spillover' into strain TA1537 (Table'5). This is also the behavior exhibited by tricyclic and larger nitroarene~ (Table 4).

4. Effects of substituents on the mutagenicity of nitroarenes Recent analytical studies of the composition of diesel emissions has indicated that they contained a large proportion of methyl-substituted nitroarenes (Riley et al., 1981; Xu et al., 1981; see also Erickson et al., 1981; Lee et al., 1980; Prater et al., 981; Schuetzle et al., 1981). Unfortunately the mutagenicity of most of these compounds has not been determined and hence the effect of methylation on the mutagenicity of the nitroarenes cannot be ascertained. This may be a major gap in our knowledge as methylation of non-nitrated polycyclic aromatic hydrocarbons has been shown to greatly affect mutagenicity [following metabolic activation (LaVoie et al., 1981a, b; Wong et al., 1981)]. The only data available on methylated nitroarenes involves methyl-substituted nitronaphthalenes (Table 4). They indicate that methylation at the C-2 position abolishes the direct-acting mutagenicity of 1-nitronaphthalene. Methylation of 2-nitronaphthalene at the C-1 position causes a diminution of the activity in strain TA98 without simultaneously affecting the activity exhibited in strain TA100. On the other hand, methylation of the molecule at the C-3 position decreases activity for TA100 while increasing that for TA98 (Table 4). These data indicate: (a) that the activity of 2-nitronaphthalene for TA98 and TA100 might be the reflection of the formation of two different types of D N A adducts, and (b) that substitution by methyl groups affects the activity of nitroarenes. Accordingly, in

235 TABLE 4 (continued) TA1538

TA98

TA98+ $9 0.2 0.01

0.8 0.06

TA1537

Reference LaVoie et al., 1981c LaVoie et al., 1981c

Roman numbers in parenthesesrefer to structure formulae.When not indicated, the structural formula of the parent arene is indicated.

view of the possible abundance of methylated nitroarenes in the environment, it would seem imperative to investigate the effect of methylation on the mutagenicity of some of the higher nitroarenes (e.g., tri- and tetracyclic nitroarenes).

5. Effects of the presence of plasmid on the mutagenicity of nitroarenes Originally, prior to the introduction of the plasmid pKM101-containing tester strains TA100 and TA98, the mutagenicity of nitroheterocyclics could not be demonstrated in the standard Salmonella mutagenicity assay (McCaUa et al., 1975; McCann et al., 1975; Tazima et al., 1975; Wang et al., 1975; Yahagi et al., 1974). Plasmid pKM101 is believed to code for error-prone DNA repair enzymes (Goze and Devoret, 1979; McCann et al., 1975; Monti-Bragadin et al., 1977; Todd et al., 1979; Walker, 1978) and it has been assumed that the ultimate mutagenic metabolite of these nitroheterocyclics reacted with the cellular DNA to form adducts that were recognized by such error-prone enzymes. With respect to the nitroarenes, the same assumption can be made as most of them either require the presence of pKM101 to express their mutagenicity (e.g., 1-nitronaphthalene and 5-nitroquinoline) in TAI00 or their mutagenicity is greatly increased by the presence of this plasmid (e.g., TA98, Table 4). It has been suggested that the carcinogenic event is also initiated by such an error-prone D N A repair process (Echols, 1981; Radman, 1977, 1980; Radman et al., 1977; Sargentini and Smith, 1981). If indeed this is so, and the carcinogenic initiation has the same chemical basis as mutagenesis in plasmid-containing strains, then an examination of the ratios of mutagenicities in the plasmid-containing strains versus the homologous non-plasmic containing parents (e.g,, TA98/TA1538) might give a clue as to the carcinogenic potential of these chemicals (Rosenkranz and Mermelstein, 1980). B. Forward mutations in Salmonella Although the mutagenicity of nitroarenes for bacteria has been most extensively studied in the reverse Salmonella mutagenicity assay developed by Ames and his associates (1975), there are several reports of the ability of these chemicals to induce forward mutations in Salmonella as well. Thus, 2-nitrofluorene has been shown to induce forward mutations to resistance to arabinose (Pueyo, 1978), and to azaguanine (Castellino et al., 1978; Bignami and Crebelli, 1979). 1.8-Dinitropyrene has been reported to induce mutations to methyltryptophan resistance (Mermelstein et al.,

24

5750 750

63 8359

0 200 50

288 0 0 0

9950 15650 1 350

85 15600 12100

2 860 1 923

338

72900 31400 7700

484 28600 36 350

2 125 860

14 471 1459

66

8 10

0 0 0

TA98

150 125 300

10 > 1000 0 0

11 800 20100 3 300

67 13400 33 000

572 186

0.04 290 195

4.8

1.8 0.04

1.7 3.0 1.6

TA1537

30 35 300

5 > 1000 0 0

0 37 6.0

0 0 303

7.3 1.0

0.2 2.8

1.6

1.8

TA1977

0 40 50

23 0

0 7.1 3.0

0 0 0

81 25

0.07 0.6 48

0

TA1978

Brown et al., 1980 Brown et al., 1980 Brown et al., 1980

McCoy et al., 1981c Brown et al., 1980 Brown et al., 1980 Brown et al., 1980

Mermelstein et al., 1981 Mermelstein et al., 1981 Mermelstein et al., 1981

Mermelstein et al., 1981 Mermelstein et al., 1981 Mermelstein et al., 1981

McCoy et al., 1981 d McCoy et al., 1981d

McCoy et al., 1981d McCoy et al., 1981d McCoy et al., 1981d

Anders et al, unpublished results

McCoy et al., 1981a Anders et al., unpublished results

McCoy et al., 1981c McCoy et al., 1981c Speck and Rosenkranz, 1980; Rosenkranz et al., 1982c

References

a BMBA, 7-bromomethyl-12-methylbenz[a]anthracene; HAQO, 4-hydroxyquinoline l-oxide. $9 derived from Aroclor-induced rat liver. * Growing cultures were used. + Qualitative tests, expressed in revertants per plate (20 #g on disc per plate). Roman numbers in parentheses refer to structural formulae. For other structures, the formulae of the parent arenes are given.

1 -Nit ro-9-aminoacridine + 3-Nitro-9-aminoacridine + 2-Nitro-9-aminoaeridine ÷

1,9-Diaminoacridine

+ 2,8-Diaminoacridine + 3,9-Diaminoacridine +

Entozon (III)

* 1,8-Dinitropyrene * 1,3,6-Trinitropyrene * 1,3,6,8-Tetranitropyrene

* 1,3-Dinitropyrene * 1,6-Dinitropyrene

* l-Nitropyrene

159 287

2,4,7-Trinitro-9-fluorenone 2,4,5,7-Tetranitro-9-fluorenone

6.5 346 1 702

16

6.1 5.9 457

29

2-Nitrofluorene 2,7-Dinitrofluorene 2,7-Dinitro-9-fluorenone

5 22

36 31

HAQO a N- Hydroxy-2-acetylaminofluorene ( + $9) BMBA a

0 0 0

TA1538

0 0 0

TAI00

Revertants per nanomole

9-Aminoacridine (I) Quinacrine (II) Proflavin

Chemical

FRAMESHIFT MUTATIONS: SPECIFICITY

TABLE 5

237

1981). In the latter case, the specific mutagenicity induced in the forward assay was of the same order of magnitude as that shown in the standard reverse Salmonella assay (Mermelstein et al., 1982). The Salmonella mutagenicity assay has, on occasion, been criticized because it is restricted; i.e., the reverse mutations in reality are a reflection of the forward mutational event. In the case of the Ames Salmonella mutagenicity assay it involves primarily in reaction with a site that is enriched with guanine a n d / o r cytosine residues. The demonstration of forward mutational activity, especially of the same order of potencies, suggests that the Salmonella tester strains in routine use at this time do not, in this instance, lead to non-representative results. C. Basis of the mutagenicity in bacteria of nitroarenes The evidence that the nitroarenes are biotransformed by bacteria to the corresponding arylhydroxylamines which in turn form adducts with the cellular D N A is compelling: (1) Although the nitroarenes induce mutations of the frameshift type, the spectrum of activities in the Salmonella tester strains suggest that this is not due to simple intercalation but rather to adduct formation. Thus, studies with D N A intercalators (proflavin, quinacrine, 9-aminoacridine, 1,9-diaminoacridine) have shown that the mutations that they induce are seen primarily, if not exclusively, in strain TA1537 and, moreover, that this activity was undiminished in strain TA1977, the uvrB ÷ analog of TA1537. This is certainly not the behavior exhibited by the majority of nitroarenes (Table 5). (2) Chemicals which induce frameshift mutations due to the formation of covalent DNA adducts either as a result of a base-displacement mechanism or because of reaction in the DNA groove are active primarily in strain TA1538 and its plasmidcontaining derivative TA98 (Table 5). This is indeed the behavior exhibited by most nitroarenes. (3) Chemicals which induce frameshift mutations as a result of such DNA adduct formation also exhibit a greatly decreased response in the uvrB ÷ analog of TA1538, i.e., TA1978 (Table 5). This is also the response elicited by nitroarenes. (4) It has already been demonstrated (see above) that nitroarenes do not react directly with purified DNA. Moreover, it has also been shown consistently that nitroarenes are primarily direct-acting mutagens in Salmonella typhimurium. In order to reconcile these apparently contradictory findings one must postulate the biotransformation of nitroarenes to biologically active intermediates. There are several lines of evidence which suggest that these are the arylhydroxylamines or their hydroxyamic acid esters. Actually, this would indicate that nitroarenes in bacteria and arylamines in mammals are converted to the same ultimate electrophilic intermediates. The former achieves this reductively while the latter do so oxidatively. T h e basis of the mutagenicity of such electrophilic intermediates has been studied in great detail in a number of laboratories. In the present context it is, therefore, significant that chemical or enzymic conversion of nitroarenes to arylhydroxylamines results in DNA-reactive intermediates as evidenced.by the effects of these intermediates on the T m values of DNA (Rosenkranz et al., 1982b, d) and their

238 ability for form DNA adducts (Howard and Beland, 1982). This is very much reminiscent of the direct DNA-modifying activity of N-naphthylhydroxylamines (Troll et al., 1963) and N-2-fluorenylhydroxylamine (Kriek, 1965; Sage and Leng, 1980; Spodheim-Maurizot et al., 1980). (5) The mutagenicity of nitroarenes has been shown to be maximal in strains deficient in the uvrB gene product (Table 5). As it has been demonstrated that the expression of the mutagenicity of DNA adduct-forming chemicals is dependent upon a deficiency in this gene product, it must be hypothesized that nitroarenes are also capable of forming covalent adducts with DNA. Contrariwise, chemicals which derive their mutagenicity from non-covalent bindings, e.g., intercalation between D N A base pairs, express their mutagenicity equally well in strains with or without a functional uvrB gene product (e.g., 9-aminoacridine in strains TA1537 and TA 1977) (Table 5). For chemicals capable of forming DNA adducts as well as of intercalating, advantage can be taken of this dichotomy in responses to estimate the contribution of each of these pathways to the mutagenicity. Thus, for Entozon (1I), intercalation (as evidenced by activity in TA1977) accounts for only a small proportion (1%) of the total mutagenicity (McCoy et al., 1981 c). (6) It has been recognized that nitro-containing chemicals, such as the nitrofurans and nitroimidazoles, are dependent on the reduction of their nitro function to the corresponding hydroxylamines for expression of mutagenicity. Indeed, bacterial strains deficient in the enzyme that converts the nitro function to the hydroxylamine (bacteria deficient in the 'classical' nitroreductase), are not susceptible to the mutagenic action of these nitro-containing chemicals (Rosenkranz and Poirier, 1979; Rosenkranz and Speck, 1975, 1976). Similarly, it was found that many nitroarenes were dependent upon this 'classical' nitroreductase to express their mutagenicity, as evidenced by their greatly decreased activity in nitroreductase-deficient bacteria (TA100NR, TA98NR, see Table 6). However, some nitroarenes express all or a major fraction of their activity even in the absence of the 'classical' nitroreductase (e.g., 1,8-dinitropyrene). Both the fact that there is residual activity expressed in TA98NR, the microorganism deficient in the 'classical' nitroreductase, and the finding of the full expression of the mutagenicity of other chemicals in TA98NR, have led to the realization that Salmonella may contain additional nitroreductases as well as other specific enzymes. Indeed, it was possible to construct (McCoy et al., 1981e; Rosenkranz et al., 1982a; Mermelstein et al., 1982) bacterial strains lacking the enzyme which recognizes 1,8-dinitropyrene (e.g., TA98/1,8-DNP 6, Table 6). Since some chemicals are fully active in both TA98NR and TA98/1,8-DNP 6 [e.g., Entozon (III) and 4-nitroquinoline 1-oxide (V)] and yet the mutagenicity of these chemicals have been shown (see McCoy et al., 1981a, c) to be dependent upon reduction of the nitro function, it is plausible that bacteria possess additional enzymes capable of reducing the nitro function. (7) That such microorganisms are indeed blocked in nitroreductase, and that this is the enzyme required for the expression of the mutagenic activity of these chemicals is evidenced by the fact that the hydroxylamine derivatives are fully active in the nitroreductase-deficient microorganism in contrast to the parent nitro compounds (Table 6).

239

(8) Additonal evidence for the existence of a nitroreductase with a specificity for 1-nitropyrene is provided by the studies of Quilliam et al. (1982) who identified, in extracts of TA100, a chromatographic peak with 1-nitropyrene-reductase activity. This peak was absent in nitroreductase-deficient TA100. Moreover, the formation of an adduct between DNA and [3H]-l-nitropyrene was also greatly reduced in ihis nitroreductase-deficient strain (QuiUiam et al., 1982). (9) If indeed it be hypothesized that the arylhydroxylamine intermediates are responsible for the mutagenicity because of their ability to react with the cellular DNA, then the increased susceptibility of resting microorganisms to the mutagenic action of nitroarenes is explicable. Arylhydroxylamines are notoriously oxygen-sensitive, being reoxidized to the nitro or nitroso compounds in the presence of oxygen (Ziegler et al., 1973) or undergoing auto-oxidation to yield the corresponding arylamine and nitrosoarene (Johnson et al., 1956; Mulvey and Waters, 1977; Lindeke et al., 1975; Weisburger and Weisburger, 1973). Facultative anaerobes, such as Salmonella typhimurium, are known to enter the fermentative stage when they are in the resting phase. Under such conditions, the arylhydroxylamines generated would be expected to be stabilized by the absence of intracellular oxygen, which in turn will account for the observed increase in mutagenicity (Table 6, and Rosenkranz and Poirier, 1979) when bacteria are incubated anaerobically. (10) The reduction of the nitro function has been thought to proceed via a nitroso intermediate. The availability of nitroreductase-deficient strains permits a determination of whether this reduction is catalyzed by a single enzyme or whether it involves a series of enzymes. Unfortunately, the available results are not clear-cut. Although as pointed out earlier (see above), the nitroreductase block can be by-passed by arylhydroxylamines (Table 6), the use of nitrosoarenes yields ambiguous results. Thus, although, as reported earlier (Ames et al., 1973), 2-nitrosofluorene is mutagenic for Salmonella (Table 6), its mutagenicity is blocked in strain TA100NR (Andrews et al., 1979) but not in TA98NR (Rosenkranz et al., 1982d; Wirth et al., 1982; Table' 6) which would indicate a dichotomy between the enzymes that catalyze the reduction of the nitro- and nitroso-functions and which could reflect the mutagenic specificity of the tester strains. On the other hand, the mutagenicity of 2-nitrosofluorene is suppressed in TA98/1,8-DNP6, the strain that is resistant to the mutagenicity not only of 2-nitrofluorene but to that of other nitroarenes as well (Table 6). It had already been established that TA98NR and TA98/1,8-DNP 6 are blocked in different functions (McCoy et al., 1982; Mermelstein et al., 1982; Rosenkranz et al., 1982a). These findings suggest that there might indeed exist two enzymes which act sequentially (Rosenkranz et al., 1982d). Results with l-nitro- and 1-nitrosopyrene (Table 6; Nilsson et al., 1981) tend to support this conclusion. Thus while 1-nitrosopyrene exhibits reduced (18% of TA98) mutagenicity in TA98NR, it is fully effective in TA98//1,8-DNP6 (130% of activity of TA98). The analogy with 2-nitro- and 2-nitrosofluorene is not, however, perfect as 1-nitropyrene (Table 6) expresses only a portion of its activity in TA98:/1,8-DNP6 (when compared to TA98). These results can be reconciled if it be hypothesized that the block in TA98/1,8-DNP6 is a specific arylhydroxylamine esterification enzyme which is required for the expression of the mutagenicity of 1,8-dinitropyrene and

383 1459 2 125

2,4,7-Trinitro-9-fluorenone

587

542

72 47

24.3

0.9 3.3

592

0.2 0.9

0.05 0.4 1.9

3-Nitro-9-fluorenone 2,7-Dinitro-9-fluorenone

- 46

0

0.65

5.2 390 126

TA98

471 2 560

- 44

- 78

- 1.8

0 0

16.1

0.11 0.86

0 0.5 I 1.8

968

4.8

T A 100NR

2,7-Dinitrofluorene

N-Hydroxy-2-aminofluorene

2-Nitrosofluorene

2-Nitrofluorene

5-Nitroquinoline 6-Nitroquinoline

0.2 0.05

15.0

N- Hydroxy-2-aminonaphthalene

1,3-Dinitronaphthalene 1,5-Dirtitronaphthalene

1.3 4.3

0.96 3.5 10.6

1 399

201

TA 100

2-Nitronaphthalene

N-Hydroxy- 1-aminonaphthalene

l -Nitronaphthalene

Niridazole (IV) 4-Nitroquinoline l-oxide (V)

Nitrofurantoin

Chemical

676

3 328

59

TA98 anaerobic

722

50 184

34 363

581

504

5 4.4

3.7

0 0

586

0.0 l 0. l I

0 0.06 2.9

17 124

0.5

TA98N R

137

79

3.9

0.36

0.2

390 106

TA98/ 1,8-DNP6

McCoy et al., 1981d

Pederson and Siak, 1981a McCoy et al., 1981 d

McCoy et al., 1981d Pederson and Siak, 1981a

Andrews et al., 1979 W a n g et al., 1980

Rosenkranz et al., 1982d Andrews et al., 1979

McCoy et al., unpublished Andrews et al., 1979 Wang et al., 1980 Pederson and Siak, 1981a

Karpinsky et al., 1982 Karpinsky et al., 1982

McCoy et al., 1981b McCoy et al., 1981b

McCoy et al., 1981b W a n g et al., 1980

McCoy et al., 1981b Nilsson et al., 1981

McCoy et al., 1981 b Nilsson et al., 1981 Rosenkranz and Poirier, 1979

Rosenkranz and Mermelstein, 1980 Mermelstein et al., 1982 McCoy et al., 1981a

Reference

M U T A G E N I C I T Y O F N I T R O A R E N E S A N D DERIVATIVE IN N I T R O R E D U C T A S E - D E F I C I E N T Salmonella typhimurium T E S T E R S T R A I N S

TABLE 6

24

21

338

0.6

< 30

2.9

0.6

< 30

60 3 568

< 1.5

< 0.6 < 0.6 55 20

30O0

3461 494

7416 3461

* Obtained by in situ reduction of 1,8-dinitropyrene (see Karpinsky et al., 1981). Results expressed as revertants per nanomole.

Entozon (1II)

4-Nitrobenzo[ g, h, i ]perylene

3-Nitroperylene

39 890

< 3 < 1.5

1-Nitrobenzo[ e ]pyrene 3-Nitrobenzo[e]pyrene

45 < 30

< 1.5

< 2.7

6-Nitrobenzo[a ]pyrene

273

1,8-Dihydroxylaminopyrene * 1,3,6-Trinitropyrene 1,3,6,8-Tetranitropyrene < 0.6 < 0.6 < 14 12

245 560 40 700 15 500

1,8-Dinitropyrene

2-Nitrochrysene 5-Nitroehrysene 6-Nitrochrysene

183 570 254000 195 774

1,6-Dinitropyrene

462 2 225

453 470 252

544 11 126

5 439 1434

4.6 0.13

144 760 54057

< 2.5

< 247 < 25

< 12 643

0.8

860

i ,3-Dinitropyrene

l-Nitrosopyrene 2-Nitropyrene

742

989 396

7-Nitrofluoranthene 8-Nitrofluoranthene

l-Nitropyrene

124 2 967

4.4

3-Nitrofluoranthene

l-Nitrofluoranthene

5-Nitroacenaphthene 9-Nitroanthracene

2,4,5,7-Tetranitro-9-fluorenone

45 890 5 840

190900 264160 176781

328

<6 60

1.6

12010 36 630 10600

319

<9 595

25 640 14000

2 750

24750 13733

1981 1981 1981 Siak, 1982a

McCoy et al., 1981c

Nilsson et al., 1981

Nilsson et al., 1981

Nilsson et al., 1981 Nilsson et al., 1981

Nilsson et al., 1981

Nilsson et al., Nilsson et al., Nilsson et al., Pederson and

McCoy et al., unpublished Rosenkranz et al., 1982c Rosenkranz et al., 1982c

Rosenkranz et al., 1982e Rosenkranz et al., 1982c Pederson and Siak, 1981a

Rosenkranz et al., 1982c Pederson and Siak, 1981a

Nilsson et al., 1981 Nilsson et al., 1981

601 494

35 35 27 83 124

Nilsson et al., 1981 Nilsson et al., 1981 Rosenkranz et al., 1982c Nilsson et al., 1981 Pederson and Siak, 1981a

198 4 945

247 2 472

Nilsson et al., 1981 Nilsson et al., 1981 Pederson and Siak, 1981a

Rosenkranz et al., 1982c Pederson and Siak, 1981a

McCoy et al., 1981d

199 371

523

0.5

1 384 370

2.7 0.07

160

242 2-nitrofluorene but not for that of 1-nitro- and 1-nitrosopyrene if 1-nitroso- or N-hydroxy-l-aminopyrene spontaneously dismutate to form the direct-action nitrenium ion (McCoy et al., 1982; Rosenkranz et al., 1982d). The above results might be relevant to the findings that while the presence of $9 overcomes in TA98NR the block to the mutagenicity of 2-nitrofluorene, 1-nitropyrene and 6-nitrochrysene, it has no such effect when T A 9 8 / 1 , 8 - D N P is the tester microorganism (Pederson and Siak, 1982a, b).

VII. Genetic activity in yeast A number of nitroarenes have been tested for their ability to induce mitotic gene conversions in Saccharomyces cerevisiae. Both 2-nitrofluorene and 2-nitronaphthalene induce such an effect in strain D3 (Simmon, 1979b). There is, however, some controversy regarding the activity of the nitropyrenes towards yeast. Thus McCoy et al. (1982b) reported that none of the nitropyrenes tested (1-nitro-, 1,3-, 1,6- and 1,8-dinitro-, 1,3,6-trinitro-and 1,3,6,8-tetranitropyrenes)had any recombinogenic activity on the yeast D4 when tested by two different protocols (the range tested was from 0.01 to 1000/lg per plate). On the other hand, Wilcox and Parry (1981) found that 1,8-dinitropyrene when tested in strains JD-1 exhibited genetic activity over a very narrow range of concentrations at which toxicity was also evident. No activity was seen either at higher or at lower concentrations. These results have led to further studies on the recombinogenic properties of the nitropyrenes. Thus a reinvestigation using several independent isolates of D4 as well as D7 and JD-1 revealed that under standard assay conditions none of the tester strains responded to the nitropyrenes (McCoy and Rosenkranz, unpublished results). Furthermore, Wilcox et al. (1982) discovered that the activity exhibited by nitropyrenes in JD-1 was observed only when the cultures were growing anaerobically. It would seem that these more recent results in effect might reconcile the various experimental findings. It may be assumed that while nitrofurans and 4-nitroquino!ine 1-oxide, both of which are recombinogenic in yeast and both of which require ~:he reduction of the nitro function, are acted upon by the equivalent of the 'classical' bacterial nitroreductase. Indeed, in Salmonella it has been shown (McCoy et al., 1981e; Mermelstein et al., 1982; Rosenkranz et al., 1982a) that the enzymes acting on nitrofurans, 4-nitroquinoline 1-oxide and nitropyrenes are not similar. If it be assumed further that the yeast lacks a nitropyrene-specific enzyme, this then may explain the non-responsiveness of the yeast to nitropyrenes. On the other hand, it is known that bacteria as well as eukaryotes possess oxygen-sensitive nitroreductases, some of these are rather non-specific, e.g., xanthine oxidases. If this is the situation, then such enzymes might be activated anaerobically and act upon nitropyrenes. In view of the demonstrated oxygen lability of hydroxylaminopyrenes, such anaerobiosis would actually by synergistic with the oxygen-sensitive enzymes.

243 VIII. Genotoxic and genetic effects of nitroarenes in cultured mammalian cells

A. Induction of DNA repair synthesis Induction of unscheduled DNA synthesis is often taken as an indication of genotoxicity and potential carcinogenicity. Martin et al. (1978) devised a modification of the assay using HeLa cells. Using such an assay, mixtures consisting of the nitration products of either pyrene, fluoranthene, perylene, chrysene, anthracene, benzo[a]pyrene, benzo[e]pyrene, benz[a]anthracene and benzo[ g, h, i]perylene were found to stimulate DNA repair synthesis (Campbell et al., 1981). It should be noted, that none of these mixtures required metabolic activation by microsomal preparations for maximal effect. Similarly, 1-nitropyrene as well as 1,3- and 1,6-dinitropyrene were reported to induced unscheduled DNA synthesis in freshly explanted human bronchus (Kawachi, 1982). 1-Nitropyrene also had such an effect on primary rat hepatocytes (Ball et al., 1982). B. Preferential inhibition of DNA repair-deficient cells Xeroderma pigmentosum cells show a preferential toxicity for a number of DNA-modifying agents (e.g., ultraviolet light, N-acetoxy-2-acetylaminofluorene, 4-nitroquinoline 1-oxide) (McCormick and Maher, 1981). Cells derived from xeroderma pigmentosum patients belonging to three different complementation groups displayed no preferential toxicity for 1,8-dinitropyrene. As a matter of fact, 1,8-dinitropyrene was not toxic for either normal or xeroderma pigmentosum cells (Arlett, 1982). C. Inhibition of DNA synthesis Exposure of cells to agents which induce DNA lesions results in an inhibition of the overall rate of DNA synthesis even after removal of the genotoxic agent. Painter and associates (Painter, 1981) have shown that using HeLa cells this could form the basis of a rapid screening test for strong mutagenic carcinogens. In that assay 2-nitrofluorene (3 × 10-3 M) gave a positive response when tested in the presence of $9 (Painter and Howard, 1982). D. Sister chromatid exchanges 2-Nitrofluorene, 1-nitropyrene and 1,8-dinitropyrene have been reported to induce moderate increases in sister-chromatid exchanges (SCE) in CHO cells when exposure was for 24 h in the absence of an $9 preparation. Addition of rat hepatic $9 resulted in greatly increased frequencies of SCEs even though exposure time was reduced to 4 h (however, see also Lewtas, 1982). In each instance, 1,8-dinitropyrene exhibited the highest activity, while 1-nitropyrene and 2-nitrofluorene had equal activities (Nachtman and Wolff, 1982). 2,4,7-Trinitro-9-fluorenone has also been reported to induce significant increases in the frequency of SCEs in CHO cells. However, in contrast to the results described above, maximal activity did not require the presence of $9. On the contrary, $9 actually caused a decrease in the incidence of SCEs induced by this chemical (Burrell et al., 1981).

244 TABLE 7 GENE MUTATIONS IN CULTURED MAMMALIAN CELLS Chemical

Response

Concentration of activity

activation

2-Nitrofluorene 2,4,7-Trinitrofluorenone

+ +

1-6 x 10 -5 M 8.7 × 10 -4 mutants/cell//~M-h/ml

Not required

l-Nitropyrene 1-Nitropyrene

+ -

< 1.0 mutants per

1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene

+ + +

74 mutants per 10 6 survivors per/tM 210 mutants per 106 survivors per/iM 152 mutants per 106 survivors per/~M

-

1,3,6-Trinitropyrene 1,3,6,8-Tetranitropyrene

+ -

65 mutants per 10 6 survivors per #M < 1.5 mutants per 106 survivors per/tM *

-

1,8-Dinitropyrene

+

0.025-2.5 ~g/ml; 48 h

1,8-Dinitropyrene

-

0.025-2.5/Lg/ml; 48 h

8-Nitroquinoline

-

1-Nitropyrene

10 6

survivors per #M *

Absent Required -

- and +

* Highest concentration tested: 20/~g per ml.

E. Clastogenicity Both 1,6- a n d 1,8-dinitropyrene i n d u c e d c h r o m o s o m a l a b e r r a t i o n s ( p r i m a r i l y a b e r r a n t m e t a p h a s e s a n d c h r o m a t i d gaps) in a d o s e - r e l a t e d fashion in a rat epithelial cell line ( R L 4 ) ( D a n f o r d et al., 1982; W i l c o x et al., 1982). The s a m e chemicals only e x h i b i t e d m a r g i n a l activity when a f i b r o b l a s t cell line of h u m a n origin was used ( W i l c o x et al., 1982). Because the R L 4 line has b e e n shown to have r e t a i n e d m e t a b o l i c e n z y m e activity ( D e a n a n d H o d s o n - W a l k e r , 1979), it is p r o b a b l e that the difference in the results b e t w e e n the two cell lines reflect their different m e t a b o l i c potentials.

F. Mutations Even t h o u g h n i t r o a r e n e s are e x t r a o r d i n a r i l y p o t e n t m u t a g e n s for Salmonella, there is a d e a r t h o f i n f o r m a t i o n r e g a r d i n g their m u t a g e n i c i t y in c u l t u r e d m a m m a l i a n cells. This is p r o b a b l y d u e to the fact that recognition of their unusual activity t o w a r d s b a c t e r i a is quite recent ( L 0 f r o t h et al., 1980; R o s e n k r a n z et al., 1980b). Still, the i n f o r m a t i o n emerging from m u t a g e n i c i t y studies with m a m m a l i a n cells c o n f i r m s the u n u s u a l b e h a v i o r of this g r o u p o f chemicals. Thus, b o t h 2 - n i t r o f l u o r e n e a n d 2,4,7-trinitro-9-fluorenone are m u t a g e n i c for m o u s e l y m p h o m a cells ( t h y m i d i n e k i n a s e locus). It w o u l d a p p e a r that n e i t h e r of these n i t r o a r e n e s requires m e t a b o l i c a c t i v a t i o n to express their m u t a g e n i c i t y ( T a b l e 7). T h e c o m p l e t e n i t r o p y r e n e series has also b e e n tested in the Chinese h a m s t e r lung f i b r o b l a s t system ( d i p h t h e r i a toxin resistance) in the a b s e n c e of m i c r o s o m a l enzymes. In that assay, while 1-nitropyrene a n d 1,3,6,8-tetranitropyrene were w i t h o u t activity, the o t h e r n i t r o p y r e n e s were m u t a g e n i c , their o r d e r of mutagenicities paralleling those r e c o r d e d for S a l m o n e l l a

245

Cell

Genetic marker

Reference

Mouse lymphoma L5178Y Mouse lymphoma L5178Y Chinese hamster CHO Mouse lymphoma L5178Y Chinese hamster lung fibroblasts Chinese hamster lung fibroblasts Chinese hamster lung fibroblasts Chinese hamster lung fibroblasts

T K +l-

Amacher et al., 1979 Burrell et al., 1981 Ball et al., 1982 Ball et al., 1982 Nakayasu et al., 1982; Kawachi, 1982

Chinese hamster lung fibroblasts Chinesen hamster lung fibroblasts

Diphtheria toxin Diphtheria toxin

TK +/TK +/Diphtheria toxin Diphtheria toxin Diphtheria toxin Diphtheria toxin

Nakayasu et al., 1982; Kawachi, 1982 Nakayasu et al., 1982; Kawachi, 1982 Nakayasu et al., 1982; Kawachi, 1982 Nakayasu et al., 1982; Kawachi, 1982 Nakayasu et al., 1982; Kawachi, 1982

Mouse lymphoma L5178YT

TG, MTX, OUA, AraC Cole et al., 1982

(Human) xeroderma pigmentosum

TG

Chinese hamster CHO

HGPRT

Arlett, 1982 Hsie, 1980

(Table 7, and Nakayasu et al., 1982; Kawachi, 1982). The presence of an $9 preparation did not result in the demonstration of the mutagenicity of 1-nitropyrene while it abolished the activity of 1,6-dinitropyrene (Nakayasu et al., 1982). On the other hand, 1-nitropyrene was non-mutagenic for Chinese hamster ovary cells (CHO) and for the mouse lymphoma system in the absence of $9. However, addition of $9 resulted in mutagenicity (Ball et al., 1982; Lewtas, 1982). Presumably in these assays exposure of the test chemicals to the biologic medium was limited to 2 - 3 h (see below). A thorough study of the mutagenicity of 1,8-dinitropyrene (in the absence of metabolic activation) in the mouse lymphoma system (thioguanine, methotrexate, ouabain and cytosine arabinoside resistances) revealed that under standard conditions (3 h of exposure) this chemical, the most potent mutagen for Salmonella, was devoid of toxicity and genetic activity. When incubation was prolonged for 24 h there still was no evidence of either toxicity or mutagenicity. However, after 48 h of incubation, even though there still was no evidence of toxicity, mutagenicity for all of the genetic endpoints became evident (Cole et al., 1982). This most unusual behavior raised a number of interesting questions: (a) What is responsible for the requirement for prolonged incubation prior to demonstration of mutagenicity: enzyme (e.g., nitroreductase) induction, the physiological state of the cells, conditioning of the medium? (b) 1,8-Dinitropyrene is a powerful frameshift mutagen for Salmonella, yet its ability to induce ouabain resistance in the mouse lymphoma cells, a locus which is taken to reflect base substitution mutations, is the same as that for the other markers tested.

246 (c) It has been pointed out by Cole et al. (1982) that this unusual behavior of 1,8-dinitropyrene raises an interesting dilemma concerning the manner of expressing mutagenic activity. If activity is expressed as mutants per survivor, one would conclude that 1,8-dinitropyrene is an extremely effective mutagen in view of the finding of 100% survival. On the other hand, expression of the activity as dose (concentration x time of exposure) would suggest that 1,8-dinitropyrene is a weak mutagen because long incubation (48 h) is required for the expression of the activity which was apparent only when fairly elevated levels of the test chemicals were used. 1,8-Dinitropyrene was also found to be devoid of mutagenic activities for human fibroblasts derived from normal individuals as well as from patients with xeroderma pigmentosum (Arlett, 1982). G. Some unexpected observations concerning genetic effects of nitroarenes on cultured mammalian cells An analysis of the results with nitroarenes in mammalian cells revealed a number of further anomalies which require elaboration. The observations of occasional requirement for exogenous metabolic activation is unusual. Although nitroarenes are potent direct-acting mutagens of Salmonella, they require, nonetheless, metabolic conversion by microbial enzymes to arylhydroxylamines or their electrophilic esters. Nitroarenes also appear to be direct-acting for Chinese hamster lung fibroblasts but exogenous metabolic activation was required for maximal ability to induce SCEs and mutations in Chinese hamster ovary cells. 2-Nitrofluorene, just as 4-nitroquinoline 1-oxide, is a direct-acting mutagen for the mouse lymphoma L5178Y system, not requiring exogenous activation and exhibiting mutagenicity within the normal response period (3 h), while 1,8-dinitropyrene required prolonged incubation. Finally, 1,8-dinitropyrene is devoid of genetic activity for normal and xeroderma human cells and also does not exhibit a preferential toxicity for xeroderma cells, even when incubation was prolonged for 48 h. Yet, mixtures of nitroarenes were reported to be direct-acting with respect to their ability to induce D N A repair synthesis in cultured human cell line (HeLa) as well as explanted human trachea (see above). Finally 1,6- and 1,8-dinitropyrene were clastogenic for cultured rat liver epithelial cells while showing negligible clastogenicity for human fibroblast cells. Obviously, a number of questions require further investigation: (a) The possible differences in these various cultured cells might reside in their metabolic capability (i.e., loss of enzymes or the nature of the metabolite that is produced); (b) the nature of the metabolite producted by the added microsomal preparations; (c) the possible differences in the DNA adduct formed by the different cells; and (d) the differences in DNA repair capabilities. H. Cell transformation Although the chemical basis of the morphological transformation (focus formation) or ability to grow in agar is generally associated with oncogenic properties, the mechanism whereby these phenomena occur has, as yet, not been elucidated. Nevertheless, the ability of nitroarenes to induce such cell transformation is included

247

herein as it is, in general, a property exhibited by genotoxic and mutagenic agents. 2-Nitrofluorene as well as 2-nitronaphthalene induced morphological transformations in Syrian hamster embryo cells. 2-Nitrofluorene also transformed baby hamster kidney ceils (BHK21). In the latter assay, maximum activity was seen in the presence of $9 preparations (Table 8). This does not, however, necessarily mean that metabolic activation is essential for transformation in the BHK21 system, as this assay responds maximally even to direct-acting alkylating agents in the presence of $9 (Purchase et al., 1978). The nitropyrenes were studied in the mouse Balb 3T3 transformation system (Sivak et al., 1981) to the limit of their solubility (Tu et al., 1982). None of them induced transformation of Balb 3T3 cells under standard conditions of the assay (Table 8). Exposure of normal human diploid fibroblasts to either 1-nitropyrene or 6-nitrobenzo[a]pyrene under anaerobic conditions resulted in a significant increase in transformants to anchorage independence. The frequency of transformants was increased dramatically when exposure was in the presence of xanthine oxidase. Transformation was abolished in the presence of oxygen (Howard et al., 1982b). Because neoplastic transformation of human diploid fibroblasts by either N-hydroxy-1- or N-hydroxy-2-naphthylamine was not dependent upon the absence of oxygen (Oldham et al., 1981), it can be concluded that the requirement for anaerobiosis was related to the bioconversion of the nitroarenes to the corresponding arylhydroxylamines by oxygen-sensitive cellular enzymes. Addition of exogenous xanthine oxidase, a nitroreductase capable of reducing nitroarenes (Howard et al., 1982a, b), enhanced the generation of the ultimate arylhydroxylamines. L Ability of 2-nitrofluorene to enhance mouse leukemia virus multiplication Carcinogens have been shown to enhance the multiplication of Moloney mouse leukemia virus-infected contact-inhibited cells. Addition of 2-nitrofluorene (10 /~g

TABLE 8 A C T I V I T Y O F N I T R O A R E N E S I~N C E L L T R A N S F O R M A T I O N ASSAYS Chemical

System

Activation required

Result

Reference

2-Nitrofluorene

H a m s t e r BHK-21 Syrian hamster embryo Syrian hamster embryo Mouse Balb 3T3 Mouse Balb 3T3 Mouse Balb 3T3 Mouse Balb 3T3 Mouse Balb 3T3 Mouse Balb 3T3

+ $9 Hamster hepatocyte None

+ +

Styles, 1981 Pienta, 1980

+

Pienta, 1980

None None None None None None

- ' -

Tu Tu Tu Tu Tu Tu

2-Nitronaphthalene 1-Nitropyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene 1,3,6-Trinitropyrene 1,3,6,8-Tetranitropyrene

et et et et et et

al., al., al., al., al., al.,

1982 1982 1982 1982 1982 1982

248

per ml) to such infected cultures resulted in a 2-fold enhancement in the yield of plaque-forming units which was taken to be at the limit of the significance of the assay (Yoshikura et al., 1979).

IX. Nature of the D N A adduct

Although, as pointed out earlier, some of the planar nitroarenes and nitroazaarenes may derived part of their mutagenicity from their ability to intercalate non-covalently between D N A base pairs, the bulk of the evidence indicates that the mutagenicity and genotoxicity of nitroarenes is due to their conversion by bacterial or mammalian enzymes to arylhydroxylamines which either react with the cellular D N A directly or following esterification to electrophilic hydroxamates. The nature of many of the adducts formed by arylhydroxylamines or their esters have been elucidated (Table 9). Mechanisms whereby these adducts cause distortion in the DNA which in turn may be responsible for their mutagenicity a n d / o r carcinogenicity have been proposed and discussed. These involve base displacement reactions or reactions result-

TABLE 9 N A T U R E OF D N A A D D U C T IDENTIFIED FOLLOWING REACTION WITH NITROARENE DERIVATIVES Arene

Adduct

References

N-Hydroxy- 1-naphthylamine

N-(Deoxyguanosin- O6-yl)- l-naphthylarnine

Kadlubar et al., 1978, 1981a; Beland, 1978 Kadlubar et al., 1978, 1981a; Beland, 1978 Kadlubar et al., 1981b

2-(Deoxyguanosin- O6-yl)- 1-naphthylamine

N-Hydroxy-2-naphthylamine

N-Hydroxy-2-acetylamino fluorene

1-(Deoxyguanosin-N2-yl)-2-naphthylamine l-(Deoxyadenosin-N6-yl)-2-naphthylamine N-(Deoxyguanosin-8-yl)-2-naphthylamine N-(Deoxyguanosin-8-yl)-2-acetylaminofluorene

N-(Deoxyguanosin-8-yl)-2-aminofluorene 3-(Deoxyguanosin-N2-yl)-2-acetylaminofluorene N-Hydroxy-2-aminofluorene

N-(Deoxyguanosin-8-yl)-2-aminofluorene

N-Hydroxy- l-aminopyrene

N-(Deoxyguanosin-8-yl)- 1-aminopyrene

Beland, 1978; Evans et al., 1980; Kriek et al., 1967; Howard et al., 1981 Westra and Visser, 1979 Sage et al., 1979; De Murcia et al., 1979; Westra et al., 1976 Westra and Visser, 1979; Beland et al., 1980; Evans et al., 1980; Spodheim-Maurizot et al, 1980 Howard et al., 1982a, b

249 ing in adduct formation in the major or minor DNA grooves, e.g., 1-hydroxylaminonaphthalene (Beland, 1978; Kadlubar et al., 1981a), N-acetoxyl-2acetylaminofluorene and its congeners (Beland, 1978; Drinkwater, et al., 1978; Evans et al., 1980; Fuchs and Daune, 1976; Fuchs et al., 1976; Grunberger and Weinstein, 1979; Levine et al., 1974; Lefevre et al., 1978; Sage and Leng, 1980; Santella et al., 1979; Yamasaki et al., 1977). Beranek and associates (1982) studied adduct formation and mutation induction in Salmonella tester strains exposed to N-hydroxy-2-aminofluorene (N-OH-2AF) and N-hydroxy-2-naphthylamine (N-OH-2-NA). Their results suggest a linear relationship between DNA adduct formation and mutagenic potency. With N-OH-2-AF only N-(deoxyguanosin-8-yl)-2-AF was identified while exposure to N-OH-2-NA resulted in the formation of N-(deoxyguanosin-8-yl)-1-(deoxyguanosin-N2-yl) - and 1-(deoxyadenosin-N6-yl)-2-NA in the ratios of 1:2:1. These findings led to the suggestion (Beranek et al., 1982) that the formation of C-8 adducts was responsible for frameshift activity while possibly the N2-deoxyguanosine adduct formed by N-OH-2-NA caused the observed base substitution activity. Of course studies with additional adducts will be required. In the case of 1,8-dinitropyrenes it was shown, however, that while it was a powerful frameshift mutagen for bacteria, it could be an effective base substitution mutagen in eukaryotic cells as evidenced by its ability to induce ouabain resistance (Cole et al., 1982).

X. Microbial metabolism of nitroarenes

Evidence has already been presented that the mutagenicity of nitroarenes for bacteria is dependent upon their enzymic conversion to arylhydroxylamines which are either direct-acting, as evidenced by their ability to react with cellular DNA e.g., naphthylhydroxylamine(Kadlubar et al., 1978, 1980) and/or hydroxylaminopyrenes (Rosenkranz et al., 1982b), or that they do so following esterification to the corresponding hydroxamates. Genetic evidence that this reduction is catalyzed by a family nitroreductases and that esterification may be controlled by a specific gene has already been presented (McCoy et al., 1981e, 1982b; Rosenkranz et al., 1982a, d). Quilliam et al. (1982) presented fractionation studies which confirmed the presence of more than one enzymic activity capable of reducing 1-nitropyrene. Moreover, these investigators also reported the isolation and identification of 1aminopyrene and N-acetyl-l-aminopyrene i n extracts of Salmonella exposed to 3H-l-nitropyrene (Quilliam et al., 1982; Messier et al., 1981). The reduction of 1-nitropyrene was very slow and was paralleled by the slow apparence of nitropyrene-DNA adducts. Formation of the DNA adducts and accumulation of 1aminopyrene in the culture medium greatly decreased in nitroreductase-deficient microorganisms. These data tend to confirm both the existence of a family of nitroreductases with specificities for the nitroarenes as well as a reduction of the nitro function and the concomitant formation of DNA adducts. These mechanisms received strong support when N-(deoxyguanosin-8-yl)-1-aminopyrene was identified as the major DNA adduct in Salmonella grown in the presence of 1-nitropyrene.

250 This is also the adduct recovered when purified DNA is exposed anaerobically to 1-nitropyrene in the presence of xanthine oxidase, a mammalian nitroreductase (Howard et al., 1982a, b). Growth of Salmonella in the presence of 1,8-dinitropyrene revealed the presence of the metabolite 1-amino-8-nitropyrene in the extract (Quilliam et al., 1982). Although the microbial biotransformation of nitroarenes has been studied primarily in Salmonella, there is evidence that other microorganisms, mainly the anaerobes, are also capable of reducing nitroarenes: 2-nitrofluorene (Karpinsky and Rosenkranz, 1980) and 1-nitropyrene (Ohnishi et al., 1981, 1982). As a matter of fact, the ability of extracts of anaerobes (Bacteroides fragilis, B. vulgatus and B. thetaiotaomicron) to active 2-nitrofluorene to a substance (presumably N-hydroxy-2aminofluorene) mutagenic in the nitroreductase-deficient strain TA98NR has been demonstrated (Karpinsky and Rosenkranz, 1980). The ability of anaerobes to activate nitroarenes is of some relevance to possible human exposures as both the colonic flora as well as the epidermal flora are rich in metabolically active anaerobes capable of performing such nitroreduction.

X|. Relationship between mutagenicity in bacteria and biotransformation by mammalian enzymes An examination of the data relating to the effects of $9 preparations on the mutagenicity of nitroarenes in Salmonella reveals a broad spectrum of responses (Table 4). These range from an abolition of the mutagenicity, to lack of any effect, to an absolute requirement for mutagenic activation. In some instances, the presence of $9 permitted expression of the mutagenicity of nitroarenes even in nitroreductase-deficient microorganisms, suggestive (Claxton et al., 1982; Kohan and Claxton, 1981; Pederson and Siak, 1981b) of the possibility that the $9 preparations contain nitroreductase activities (see also Speck and Rosenkranz, 1975, 1976; Rosenkranz and Poirier, 1979). A study of the S9-mediated inactivation of 1,8-dinitropyrene revealed that this inactivating activity was microsome-associated (e.g., sedimentable at 100000 × g), heat-sensitive, and required NADP. It was present not only in rat hepatic $9 but also in the $9 prepared from mouse and hamster livers. The activity was maximal if the preparations were derived from Aroclor-induced animals (Mermelstein et al., 1982; Rosenkranz et al., 1982b; see also Pederson and Siak, 1982b). A number of explanations for these effects of $9 on the expression of the mutagenicity may be offered: (1) It is known that bacterial nitroreductases do not possess and unlimited specificity for nitroarenes. Thus, it was shown previously that 8-nitroquinoline as well as 1,8-dinitronaphthalene, which are non-mutagenic for Salmonella per se, could be rendered mutagenic following chemical reduction of the nitro function to the corresponding hydroxylamines (Karpinsky et al., 1982). Similarly, as it is known that $9 contains nitroreductase activity, it is conceivable that some of the test chemicals which require $9 for the demonstration of their mutagenicity do so

251 because their nitro functions are refractory to reduction by the bacterial nitroreductase but that this biotransformation is accomplished by the nitroreductases present in the $9 preparation. (2) In a number of instances in which the in vitro metabolism of nitroarenes was studied, only the corresponding arylamine was detected (Poirier and Weisburger, 1974). No evidence of hydroxylamine intermediates was obtained. There are several possible explanations for such an observation. (a) The hydroxylamines are very unstable, especially in the presence of oxygen, and hence, due to their transient character they could not be detected (see above). (b) The enzymic conversion of arylhydroxylamines to arylamines is very rapid (Poirier and Weisburger, 1974) and hence, the intermediate may not be detected. (c) The arylhydroxylamine intermediate is most unstable and can autooxidize to nitrosoarene and to the corresponding arylamine (see above). (d) Although it is hypothesized that the reduction of nitroarenes proceeds via the nitroso and hydroxylamine intermediates, these moieties may be enzyme-bound (McCoy et al., 1981a) and hence, not available biologically until fully reduced to the arylamine state. (e) If indeed the nitroarenes are reduced by mammalian enzymes all the way to arylamines, and since the arylamines are not direct-aging mutagens, the possibility exists that the mutagenicity of these chemicals can be explained by their reoxidation by cytochrome P450-dependent enzymes to the arylhydroxylamines. However, it must be noted that generally arylamines exhibit a much lower mutagenicity, in the presence of $9, than the corresponding nitroarenes in the absence of $9. Moreover, such an S9-catalyzed effect cannot explain the extent of the mutagenicity observed when $9 is added to nitroarenes to overcome their nitroreductase deficiency in indicator strains TA98NR. The same comment applies to the observed mutagenicity of some of the nitroarenes that do not require $9, especially when compared to the mutagenicity of the corresponding arylamines. (3) The requirement of the $9 may not be related, necessarily, to the biotransformation of the nitro function but rather to ring oxydation. Thus, studies (E1-Bayoumy and Hecht, 1981, 1982) of the S9-induced products of 5-nitroacenaphthene have shown the presence of the major metabolites, 1-hydroxy-5-nitro- and 2-hydroxy-5nitroacenaphthene. On the basis of the kinetics of their appearance and their mutagenicity in the presence and absence of $9, EI-Bayoumy and Hecht (1982) propose that the hydroxy- and oxo-5-nitroacenaphthenes are the proximate mutagens which are then reduced further to the nitroso- or hydroxylamino-acenaphthenes. Further metabolism leads to 1,2-dihydroxy-5-nitroacenaphthene and to the aminonaphthene derivatives which are viewed as detoxification products. These studies indicate, therefore, that the initial major route of metabolic activation of 5-nitroacenaphthene is C-hydroxylation presumably followed by nitroreduction. Examination of the S9-catalyzed metabolism of l-nitronaphthalene revealed the presence of hydroxy and dihydrodiol derivatives of 1-nitronaphthalene. Neither N-hydroxy-l-aminonaphthalene nor 1-aminonaphthalene were detected. This also suggests detoxification of 1-nitronaphthalene via the aromatic C-hydroxylation. No major nitroreduction was observed (E1-Bayoumy and Hecht, 1981).

252

Studies on the metabolism of 6-nitrobenzo[a]pyrene, a chemical which requires $9 to express its mutagenicity in Salmonella (Table 4), revealed the presence of a series of intermediates (1-hydroxy- and 3-hydroxy-6-nitrobenzo[a]pyrene, 6-nitrobenzo[a]pyrene-l,9- and -3,9-hydroxyquinone) which still required $9 to express their mutagenicity (Fu et al., 1981, 1982; see also Tong et al., 1982). The studies by Pitts et al. (1982b) have revealed that the S9-induced mutagenicity of 6nitrobenzo[a]pyrene by-passes the nitroreductase block in the nitroreductase-deficient strain TA98NR (see also Pederson and Siak, 1982a). It may, therefore, well be asked whether indeed the mutagenicity of the metabolite of 6-nitrobenzo[a]pyrene involves reduction of the nitro function or simply adduct formation between DNA and the oxygenated benzo[a]pyrene moiety, in analogy with the action of benzo[a]pyrenediolepoxide. Moreover, the desnitrobenzo[a]pyrene-3,6-quinone has been detected in incubation mixtures supplemented with $9 (Fu et al., 1982). Furthermore dihydroxy derivatives of 6-nitrobenzo[a]pyrene were also detected when the latter was exposed either to intact hamster embryonic fibroblasts or to rat liver $9 (Tong et al., 1982). However, in view of the demonstrated substrate specificity of the bacterial nitroreductases, it is also conceivable that the mutagenicity of the oxygenated metabolite of 6-nitrobenzo[a]pyrene does require reduction of the nitro function but that this reduction is not carried out by the 'classical' nitroreductase which is the enzyme lacking in TA98NR. It might well involve a different nitroreductase. This possibility requires further investigation, especially in view of the observation (Pederson and Siak, 1982a, b) that while incubation of some nitroarenes (e.g., 2-nitrofluorene, 1-nitropyrene, 6-nitrochrysene) in the presence of $9 overcomes the block in TA98NR, the microorganism deficient in the 'classical' nitroreductase, it does not overcome the block in TA98/1,8-DNP 6, the microorganism deficient in another enzyme with a specificity for nitroarenes (McCoy et al., 1981e). This may reflect the possibility that ring-oxidized nitroarenes exhibit a different substrate specificity towards TA98/1,8-DNP 6, than the parent nitroarene. Alternatively, it was recently shown (Rosenkranz et al., 1982d) that the 'classical' nitroreductase (i.e., deficient in TA98NR) and TA98/1,8-DNP 6 differ in substrate recognition towards nitrosoarenes and hydroxylaminoarenes and that the lesion in T A 9 8 / 1 , 8 - D N P 6 involves primarily esterification of the arylhydroxylamine. It can then by hypothesized that the nitroreductase present in $9 permits by-passing of the block in TA98NR but it does not supply the activity missing in TA98/I,8-DNP 6 (McCoy et al., 1982a). Preliminary studies with 1-nitropyrene also support the possibility that two activation pathways might be involved. Thus 1-amino- and N-acetylamino pyrene have been detected upon incubation of this nitroarene with $9 (Ball et al., 1982). This is consistent with reduction of the nitro function and with the differential effects of 1-nitropyrene and $9 in TA98NR and TA98/1,8-DNP 6 (see above). In addition, similarly to the findings with 6-nitrobenzo[a]pyrene (Fu et al., 1981), ring hydroxylation of the pyrene moiety has also been reported (Ball et al., 1982; Pederson and Siak, 1982). In a recent preliminary report EI-Bayoumy et al. (1982) tentatively identified 4,5-dihydro-4,5-dihydroxy-l-nitropyrene and two monohydroxy nitropyrenes when

253 1-nitropyrene was incubated with $9 aerobically. 1-Aminopyrene was not detected. When the reaction was carried out under reduced oxygen tension (4% in nitrogen), extensive reduction to 1-aminopyrene was observed. Similarly, incubation of 6nitrochrysene under aerobic conditions with $9 resulted in generation of an angular 6-nitrochrysene dihydrodiol without the detection of 6-aminochrysene. When the oxygen level was lowered, the yield of the nitrodihydrodiol decreased and 6aminochrysene was observed (EI-Bayoumy et al., 1982). These various observations might indeed explain the S9-mediated by-passing of the block in TA98NR.

XII. Biotransformation of nitroarenes in animals

Intraperitoneal administration of non-radioactive 1-nitronaphthalene and 2nitronaphthalene to rats has been reported to result in the urinary excretion of the corresponding aminonaphthalenes (Johnson and Cornish, 1978). No other naphthalene derivatives were detected. On the other hand, oral intubation of the rat with [3H]-2-nitronaphthalene resulted in the urinary excretion of N-hydroxy-2aminonaphthalene and its glucuron!de (Kadlubar et al., 1981b) as well as of 2-amino-l-naphthol. Further studies revealed that these urinary N hydroxynaphthalene-N-glucuronides are rapidly hydrolyzed at the acidic pH of the urine and are, therefore, accessible to the bladder epithelium where they can be resorbed into the circulation. It was suggested that at the acidic pH of urine (pH 5) these extracellular N-hydroxyarylamines are converted to electrophilic arylnitronium ion-carbocation ions which upon passage through the urothelium are capable of forming DNA adducts and thus of initiating the neoplastic process (Olgesby et al., 1981; see also Kadlubar et al., 1977). Similarly, N-hydroxy-2-aminonaphthalene (and 2-nitrosonaphthalene) was also isolated from the urines of monkeys but not of dogs that received 2-nitronaphthalene (Radomski et al., 1973). Although traces of 2-aminonaphthalene were detected in the urine of monkeys, none was detected in dog urine. The lack of detection of 2-aminonaphthalene in these studies probably reflects the different routes of administration (intraperitoneal versus intubation) and species differences in the formation of the hydroxylamine derivative. These studies confirm that N-hydroxylaminonaphthalene is the active or penultimate metabolite and they suggest that the detoxification of 2-nitronaphthalene proceeds via 2-amino-l-naphthol (Kadlubar et al., 1981b).

XIII. Studies with whole animals

A. Induction of sperm-head abnormalities Induction of sperm abnormalities has been taken as an indication of mutagenic and carcinogenic potentials (Wyrobeck and Bruce, 1978; Topham, 1980). Intraperitoneal administration of 2-nitrofluorene to mice (205-1500 m g / k g / d a y for 5 days) did not result in the induction of sperm-head abnormalities (Topham, 1980).

Mouse

6-Nitrofluoranthene

Skin + promotion

Skin + promotion Subcutaneous

Mouse Rat

3-Nitrofluoranthene

2-Nitronaphthatene 2-Nitrofluorene 1-Nitropyrene

l-Nitronaphthalene

Feed Feed Feed Feed Feed Subcutaneous

Rat, mouse Rat, mouse, hamster Rat, mouse Dog, monkey Rat Rat

5-Nitroacenaphthene

Administration

Species

Chemical

TABLE 10 C A R C I N O G E N I C I T Y OF N I T R O A R E N E S

Distal tumors Distal tumors Negative Bladder tumors Distal tumors Sarcomas (histiocytomas) at site of injection Negative Sarcomas (histiocytomas) at site of injection Skin tumors

Result

EI-Bayoumy et al., 1982

EI-Bayoumy et al., 1982 Ohgaki et al., 1982

Griesemer and Cuoto, 1980 IARC, 1978 Griesemer and Cuoto, 1980 Poirier and Weisburger, 1979 Weisburger and Weisburger, 1958 Ohgaki et al., 1982

Reference

ix

255 Topham (1980) made the observation that there was a correlation between the ability of a chemical to induce sperm-head abnormalities and its capacity of inducing transmissible genetic changes in the whole animal (e.g., specific-locus mutations, heritable translocations and dominant lethals) but not between induction of sperm abnormalities and mutagenicity in bacteria or carcinogenicity in animals. According to Topham, therefore, the present findings suggest, by analogy, that 2-nitrofluorene will not be found to be an inducer of germ cell mutations in mammals. Unfortunately, there is no data available in the literature dealing with the mutagenic potential of nitrofluorene for whole animals to elucidate this possibility. B. Host-mediated assay The host-mediated assay was originally devised such that metabolic activation of chemicals requiring biotransformation to express their mutagenicity would be carried out by the whole animal. To achieve this, the animal receives the indicator microorganism, e.g., Salmonella or yeast, at one site (intraperitoneal injection) while the test chemical is administered either intramuscularly or by oral intubation. Using direct-acting mutagens in such assays may introduce a number of artifacts as reactive species may bind to cellular constituents at the side of injection and not reach the target microorganisms. Similarly, other chemicals, such as the nitroarenes, might be biotransformed in the liver to inactive or less active metabolites prior to contact with their microorganism. With respect to the nitroarenes, as previously shown (see above) both 2nitronaphthalene and 2-nitrofluorene are mutagenic for Salmonella and recombinogenic for the yeast Saccharomyces cerevisiae D3. In the host-mediated assay, however, these activities were either absent or dependent upon the route of administration. Thus, 2-nitrofluorene when given intramuscularly (125 mg per kg) or by oral intubation (1600 mg per kg) was mutagenic for Salmonella TA1538; however, with Saccharomyces cerevisiae D3, even 1600 mg per kg administered by oral intubation was not recombinogenic (Simmon et al., 1979). 2-Nitronaphthalene, in the host-mediated assay, was mutagenic for Salmonella TA1530 and TA1538 when administered intramuscularly (125 mg per kg), but delivery by oral intubation (1300 mg per kg) did not prove mutagenic for Salmonella typhimurium nor recombinogenic for Saccharomyces cerevisiae.

XIV. Carcinogenicity in animals

A search of the literature revealed information on the carcinogenicity of 6 nitroarenes (Table 10); 5-nitroacenaphthene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 3-nitrofluoranthene and 2-nitrofluorene. With the exception of 1-nitronaphthalene, all of the nitroarenes tested were carcinogenic. There is insufficient data as yet to make any generalization concerning the relationship of mutagenic potency to the potential for causing cancer, except that it might be pointed out that a comparison of the mutagenic potencies of'5 of these chemicals obtained in a single laboratory (McCoy and Rosenkranz et al., see Table 4) revealed that 1-

256 nitronaphthalene exhibited the lowest mutagenicity. A preliminary report also indicates that while 6-nitrochrysene was endowed with tumor-initiating activity, such a property was not exhibited by 1-nitropyrene (1 mg nitroarene followed by 13 weeks of promoter treatment; E1-Bayoumy et al., 1982).

XV. Some thoughts on nitroarenes

It has been calculated (Rosenkranz, 1982) that the annual emission rate of 1-nitropyrene from light diesel passenger cars in the United States will be 15 000 kg by 1990. This is, of course, a low level when compared to the industrial production of the related nitrobenzenes and nitrotoluenes, which are in the range of millions of kg per year (Hartter, 1982). Still, while the nitro derivatives of monocyclic aromatic hydrocarbons are produced in an industrial environment and are mostly contained therein, the related nitrated polycyclic aromatic hydrocarbons are emitted into the environment mainly in the form of combustion products. Their potent mutagenicity and the fact that the most abundant of these, 1-nitropyrene (one of the nitroarenes with a relatively low mutagenic potency), had been shown to be carcinogenic for rodents, should warn us of a possible hazard. Because of the foreseen dieselization of the passenger car (Ingalls and Bradow, 1981), the nitroarenes will continue to present us with a problem. In addition, it has recently been found that some of the newer energy technologies also lead to the formation of nitroarenes. Thus it would seem that a better understanding of the properties of these chemicals and whether they are indeed bioavailable following inhalation (the major expected route of exposure) is urgently needed. There is evidence that while these chemicals are readily desorbed under physiological conditions from diesel particulate material (King et al., 1981), such does not appear to be the case for other combustion products containing nitroarenes, e.g., fly ash (Mosberg et al., 1981) and carbon blacks (Agurell and L~froth, 1982; Giammarise et al., 1982; Rosenkranz et al., 1980b). If indeed the nitroarenes cause a problem to man and to this environment, then it is fortunate that the formation of the nitroarenes in the diesel engine is not essential to its operation. Apparently, they are formed in the after-burn, due to the coincidental presence of polycyclic aromatic hydrocarbons, oxides of nitrogen and traces of acid. Since the technology appears to be available for eliminating or greatly reducing their presence in the emitted particulates, while uncertainties persist regarding their biologic effects, the prudent course of action appears to be to use such technology.

References

Agurell, E., and G. L6froth (1982) Presence of various types of mutagenic impurities in carbon black detected by the Salmonella/microsome assay, in: M.D. Waters et al. (Eds.), Short-Term Bioassaysin the Analysis of Complex Environmental Mixtures, III. Plenum, New York, in press. Amacher, D.E., S.C. Paillet and G.N. Turner (1979) Utility of the mouse lymphoma L5178Y/TK assay for the detection of chemical mutagens, in: A.W. Hsie, J.P. O'Neill and V.K. McElheny (Eds.), Banbury Report No. 2: Mammalian Cell Mutagenesis: The Maturation of Test Systems, Cold Spring Harbor Laboratory, New York, pp. 277-289.

257 Ames, B.N., F.D. Lee and W.E. Durston (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70, 782-786. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31,347-364. Anders, M., G.E., Karpinsky, E.C. McCoy and H.S. Rosenkranz (1982) Phenotypic instability of Salmonella typhimurium tester strains: An example of a plasmid-enhanced genetic drift? Mutation Res., 97, 411-428. Andrews, L.S., L.R. Pohl, J.A. Hinson and J.R. Gillette (1979) Mutagenesis of 2-nitrofluorene (NF), 2-nitrosofluorene (NOF) and 2-hydroxylaminofluorene (NHOHF) for Salmonella TAI00 and TA100FR, Eighteenth Annual Meeting, Scoiety of Toxicology, Abstract A48. Arlett, C. (1982) Mutagenicity of 1,8-dinitropyrene in mammalian celts,in: E.C. Rickert (Ed.), The Toxicity of Nitroaromatic Compounds, Hemisphere, New York, in press. Ball, L.M., M.J. Kohan, L. Claxton and J. Lewtas (1982) Mammalian activation and metabolism of 1-nitropyrene, Abstracts, Fifth CIIT (Chemical Industry Institute of Toxicology) Conference on Toxicology: Toxicity of Nitroaromatic Compounds, p. 6. Beland, F.A. (1978) Computer-generated graphic models of the N Z-substituted deoxyguanosine adducts of 2-acetylaminofluorene and benzol a ]pyrene and the O6-substituted deoxyguanosine adduct of 1-naphthylamine in the DNA double helix, Chem.-Biol. Interact., 22, 329-339. Beland, F.A., W.T. Allaben and F.E. Evans (1980) Aryl-transferase mediated binding of N-hydroxyarylamide to nucleic acids, Cancer Res., 40, 751-757. Beranek, D.T., R.H. Heflich, F.A. Beland, F.F. Kadlubar and G.L. White (1982) The relationship between arylamine-DNA binding and mutagenicity in Salmonella typhimurium, Environ. Mutagen., 4, 297-298. Bignami, M., and R. Crebelli (1979) A simplified method for the induction of 8-azaguanine resistance in Salmonella typhimurium, Toxicol. Lett., 3, 169-175. Brafia, M.F., J.M. Castellano, C.M. Roldan, A. Santos, D. Vazquez and A. Jimenez (1980) Synthesis and mode(s) of action of a new series of imide derivatives of 3-nitro-l,8-naphthalic acid, Cancer Chemother. Pharmacol., 4, 61-66. Brown, B.R., W.J. Firth III and L.W. Yielding (1980) Acridine structure correlated with mutagenic activity in Salmonella, Mutation Res., 72, 373-388. Brusick, D., V.F. Simmon, H.S. Rosenkranz, V. Ray and R.S. Stafford (1980) An evaluation of the E. coli WP2 and uvrA reverse mutation assay, Mutation Res., 76, 169-190. Burrell, A.D., J.J. Andersen, M.M. Jotz, E.L. Evans and A.D. Mitchell (1981) Genetic toxicity of 2,4,7-trinitrofluoren-9-one in the Salmonella assay, L5178Y TK +/- mouse lymphoma mutagenesis assay and sister chromatid exchange assay, Environ. Mutagen., 3, 360. Campbell, J., G.C. Crumplin, J.V. Garner, R.C. Garner, C.N. Martin and A. Rutter (1981) Nitrated polycyclic aromatic hydrocarbons: potent bacterial mutagens and stimulators of DNA repair synthesis in cultured human cells, Carcinogenesis, 2, 559-565. Castellino, S., R. Lorenzetti, L. Ravenna and I. de Carneri (1978) Forward mutation assays using bacteria and 8-azaguanine, Ecotoxicol. Environ. Safety, 2, 277-299. Cheli, C., D. deFrancesco, L.A. Petrullo, E.C. McCoy and H.S. Rosenkranz (1980) The Salmonella mutagenicity assay: Reproducibility, Mutation Res., 74, 145-150. Chiu, C.W., L.H. Lee, C.Y. Wang and G.T. Bryan (1978) Mutagenieity of some commercially available nitro compounds for Salmonella typhimurium, Mutation Res., 58, 11-22. Clark, D.R., A.L. Brooks, A.P. Li, W.M. Hadley, R.L. Hanson and R.O. McClelland (1982) Mutagenicity of fossil fuel combustion products in standard and nitroreductase-deficient Salmonella typhimurium, Environ. Mutagen., 4, 333-334. Claxton, L.D., M. Kohn and J. Lewtas (1982) A comparison of the effect of metabolizing factors upon the mutagenicity of two nitroarene compounds and diesel exhaust organics, Environ. Mutagen., 4, 382. Cole, J., C.F. Arlett, J. Lowe and B.A. Bridges (1982) The mutagenic potency of 1,8-dinitropyrene in cultured mouse lymphoma ceils, Mutation Res., 93, 213-220. Danford, N., P. Wilcox and J.M. Parry (1982) The clastogenic activity of dinitropyrenes in a rat liver epithelial cell line, Mutation Res., 105, 349-355.

258 Dean, B.J., and G. Hodson-Walker (1979) An in vivo chromosome assay using cultured rat liver cells, Mutation Res., 64, 329--337. De Murcia, G., M.-C.E. Lang, A.-M. Freund, R.P.P. Fuchs, M.P. Daune, E. Sage and M. Leng (1979) Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in colE1 DNA by means of specific antibodies, Proc. Natl. Acad. Sci. (U.S.A.), 76, 6076-6080. De Serres, F.J., and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Mutation Res., 64, 159-165. Drinkwater, N.P., J.A. Miller, E.C. Miller and N.C. Yang (1978) Covalent intercalative binding to DNA in relation to the mutagenicity of hydrocarbon epoxides and N-acetoxy-2-acetylarninofluorene, Cancer Res., 38, 3247-3255. Dunkel, V.C., and V.F. Simmon (1980) Mutagenic activity of chemicals previously tested for carcinogenicity in the National Cancer Institute Bioassay Program. in: R. Montesano, H. Bartsch and L. Tomatis (Eds.), Molecular and Cellular Aspects of Carcinogenic Screening Tests, IARC Scientific Publication No. 27, International Agency for Research on Cancer, Lyon, pp. 283-301. Echols, H. (1981) SOS functions, cancer and inducible evolution, Cell, 25, 1-2. E1-Bayoumy, K., and S.S. Hect (1981) Comparative metabolism of nitropolynuclear aromatic hydrocarbons, in: Sixth International Symposium on Polynuclear Aromatic Hydrocarbons, Abstracts, Battelle Laboratories, Columbus, Ohio. EI-Bayoumy, K., and S.S. Hecht (1982) Identification of mutagenic metabolites formed by C-hydroxylation and nitroreduction of 5-nitroacenaphthene in rat liver, Cancer Res., 42, 1243-1248. El-Bayoumy, K., E.J. Lavoie, S.S. Hecht, E.A. Fow and D. Hoffmann (1981) The influence of methyl substitution on the mutagenicity of nitronaphthalenes and nitrobiphenyls, Mutation Res., 81, 143-153. El-Bayoumy, K., S.S. Hecht and E.L. Wynder (1982) Tumor-initiating activity and in vitro metabolism of 6-nitrochrysene and l-nitropyrene, Proc. Am. Assoc. Cancer Res., 23, 84. Epler, J.L., W. Winton, T. Ho, F.W. Larimer, T.K. Rao and A.A. Hardigree (1977) Comparative mutagenesis of quinolines, Mutation Res., 39, 285-296. Erickson, M.D., D.L. Newton, M.C. Saylor, K.B. Tomer, E.D. Pellizzari, R.B. Zweidinger and S. Tejada (1981) Fractionation and identification of organic components in diesel exhaust particulate, EPA Diesel Emissions Symposium Abstract. Evans, F.E., D.W. Miller and F.A. Beland (1980) Sensitivity of the conformation of deoxyguanosine to binding at the C-8 position by N-acetylated and unacetylated 2-aminofluorene, Carcinogenesis, 1, 955-959. Fu, P.P., M.W. Chou, L.E. Unruh, F.A. Beland, F.F. Kadlubar, D.A. Casciano, R.H. Heflich and F.E. Evans (1983) In vitro metabolism of 6-nitrobenzo[a]pyrene: Identification and mutagenicity of the metabolites, in: Sixth International Symposium Polynuclear Aromatic Hydrocarbons, Abstracts, Battelle Laboratories, Columbus, Ohio. Fu, P.P., M.W. Chou, S.K. Yang, F.A. Beland, F.F. Kadhibar, D.A. Casciano, R.H. Heflich and F.E. Evans (1982) Metabolism of the mutagenic environmental pollutant, 6-nitrobenzo[a]pyrene: Metabolic activation via ring oxidation, Biochem. Biophys. Res. Commun., 150, 1037-~1043. Fuchs, R., and M. Daune (1976) Physical basis of chemical carcinogenesis by N-2-fluorenylacetamide derivatives and analogs, FEBS Lett., 34, 295-298. Fuchs, R.P.P., J.-F. Lefevre, J. Panyet and M.P. Daune (1976) Comparative orientation of the fluorene residue in native DNA modified by N-acetoxy-N-2-acetylaminofluorene and two 7-halogeno derivatives, Biochem., 15, 3347-3351. Fukino, H., S. Mimura, K. Inoue and Y. Yamane (1982) Mutagenicity of airborne particles, Mutation Res., 102, 237-247. Gajcy, H., B. Chlopkiewicz and J. Koziorowska (1979) Comparative study of mutagenic, inductive and transforming activities of Ledakrin, Pol. J. Pharmacol. Pharm., 31, 661-666. Giammarise, A.T., D.L. Evans, M.A. Butler, C.B. Murphy, D.K. Kiriazides, D. Marsh and R. Mermelstein (1982) Improved methodology for carbon black extraction, in: Sixth International Symposium on Polynuclear Aromatic Hydrocarbons, Battelle Laboratories, Columbus, Ohio, pp. 325-334. Gibson, T.L. (1981) Nitro derivatives of polynuclear aromatic hydrocarbons in airborne and source particulate, EPA Diesel Emissions Symposium Abstracts.

259 Goze, A., and R. Devoret (1979) Repair promoted by plasmid pKM101 is different from SOS repair, Mutation Res., 61, 163-179. Green, M.H.L., and W.J. Muriel (1976) Mutagen testing using trp + reversion in Escherichia coli, Mutation Res., 38, 3-32. Griesemer, R.A., and C. Cueto Jr. (1980) Toward a classification scheme for degrees of experimental evidence for the carcinogenicity of chemicals for animals, in: R. Montesano, H. Bartsch and L. Tomatis (Eds.), Molecular and Cellular Aspects of Carcinogen Screening Tests, IARC Scientific Publication No. 27, International Agency for Research on Cancer, Lyon, pp. 259-281. Grunberger, D., and I.B. Weinstein (1979) Conformational changes in nucleic acids modified by chemical carcinogens, in: P.L. Grover (Eds.), Chemical Carcinogens and DNA, Vol. II, CRC Press, Boca Raton, Florida, pp. 59-63. Hartter, D.R. (1982) The use and importance of nitroaromatic compounds in the chemical industry, in: D.E. Rickert (Ed.), The Toxicity of Nitroaromatic Compounds, Hemisphere, New York, in press. Henderson, T.R., J.D. Sun, R.E. Royer, C.R. Clark, T.M. Harvey, D.F. Hung, J.E. Fulford, A.M. Lovett and W.R. Davidson (1981) G C / M S and MS/MS studies of direct-acting mutagens in diesel emissions, EPA Diesel Emissions Symposium Abstract. Ho, Y.L., and S.K. Ho (1981) Screening of carcinogens with the prophage clts857 induction test, Cancer Res., 41,532-536. Ho, C.-H., B.R. Clark, M.R. Guerin, B.D. Barkenbus, T.K. Rao and J.L. Epler (1981) Analytical and biological analyses of test materials from the synthetic fuel technologies, IV. Studies of chemical structure-mutagenic activity relationships of aromatic nitrogen compounds relevant to synfuels, Mutation Res., 85, 335-345. Howard, P.C., and F.A. Beland (1982) Xanthine oxidase catalyzed binding of 1-nitropyrene to DNA, Biochem. Biophys. Res. Commun., 104, 727-732. Howard, P.C., D.A. Casciano, F.A. Beland and J.G. Shaddock, Jr. (1981) The binding of N-hydroxy-2acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures, Carcinogenesis, 2, 97-102. Howard, P.C., F.A. Beland, F.E. Evans and R.H. Heflich (1982a) Xanthine oxidase-catalyzed reduction of l-nitropyrene to a potentially mutagenic DNA-binding species, Proc. Am. Assoc. Cancer Res., 23, 62. Howard, P.C., F.A. Beland, F.E. Evans, R.H. Heflieh, G.E. Milo and F.F. Kadlubar (1982b), l-Nitropyrene: In vitro metabolism and DNA adduct formation, in: D.E. Rickert (Ed.), The Toxicity of Nitroaromatic Compounds, Hemisphere, New York, in press. Hsie, A.W. (1980) Quantitative mutagenesis and mutagen screening with Chinese hamster ovary cells, in: G.M. Williams, R. Kroes, H.W. Waaijers and K.W. van de Poll (Eds.), The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier/North-Holland, Amsterdam, pp. 89-102. IARC (1978) IARC Monograph on the Evaluation of the Carc'mogenic Risk of Chemicals to Humans: Some Aromatic Amines and Related Nitro Compounds; Hair Dyes, Colouring Agents and Miscellaneous Industrial Chemicals, Vol. 16, International Agency for Research on Cancer, Lyon, France. Ingalls, M.N., and R.L. Bradow (1981) Particulate trends with increased dieselization, Air Poll. Control Assoc. Abstract, 74th Annual Meeting, 81-56.2. J~iger, J. (1978) Detection and characterization of nitro derivatives of some polycyclic aromatic hydrocarbons by fluorescence quenching after thin-layer chromatography: Application to air pollution analysis, J. Chromatogr., 152, 573-578. Jin, Z.L., X.B. Xu, J.P. Nachtman and E.T. Wei (1982) Potent mutagenic impurities in a commercial sample of 3-nitro-9-fluorenone, Cancer Lett., 15, 209-214. Johnson, D.E., and H.H. Cornish (1978) Metabolic conversion of 1- and 2-nitronaphthalene to 1- and 2-naphthylamine in the rat, Toxicol. Appl. Pharmacol., 46, 549-553. Johnson, D.H., M.A.T. Rogers and G. Trappe (1956) Aliphatic hydroxylamines, Part II, Autoxidation, J. Chem. Soc., 1093. Jungers, R.H., and J.E. Bumgarner (1981) Diesel bus terminal study; Characterization of volatile and particle bound organics, EPA Diesel Emissions Symposium Abstracts. Kadlubar, F.F., J.A. Miller and E.C. Miller (1977) Hepatic microsomal N-ghicuronidation and nucleic

260 acid binding of N-hydroxy arylamines in relation to urinary bladder carcinogenesis, Cancer Res.. 37, 805 - 814. Kadlubar, F.F., J.A. Miller and E.C. Miller (1978) Guanyl O6-arylamidation and O6-arylation of DNA by the carcinogen N-hydroxy-1-naphthylamine, Cancer Res., 38, 3628-3638. Kadlubar, F.F., L.E. Unruh, F.A. Beland, K.M. Straub and F.E. Evans (1980) In vitro reaction of the carcinogen, N-hydroxy-2-naphthylamine, with DNA at the C-8 and N 2 atoms of guanine and at the N 6 atom of adenine, Carcinogenesis, 1, 139-150. Kadlubar, F.F., W.B. Melchior Jr., TJ. Flammang, A.G. Gagliano, H. Yoshida and N.E. Geacintov (1981a) Structural consequences of modification of the oxygen atom of guanine in DNA by the carcinogen N-hydroxy-l-naphthylamine, Cancer Res., 41, 2168-2174. Kadlubar, F.F., L.E. Unruh, T.J. Flammang, D. Sparks, R.K. Mitchum and G J . Mulder (1981b) Alteration of urinary levels of the carcinogen, N-hydroxy-2-naphthylamine, and its N-glucuronide in the rat by control of urinary pH, inhibition of metabolic sulfation, and changes in biliary excretion, Chem.-Biol. Interact., 33, 129-147. Kalinowska, E., and M. Chorazy (1980) Mutagenic activity of some 9-aminoalkyl acridine derivatives on S. typhimurium, Mutation Res., 78, 7-15. Karpinsky, G.E., and H.S. Rosenkranz (1980) The anaerobe-mediated mutagenicity of 2-nitrofluorene and 2-aminofluorene for Salmonella typhimurium, Environ. Mutagen., 2, 353-358. Karpinsky, G.E., E.C. McCoy, H.S. Rosenkranz and R. Mermelstein (1982) The chemical activation of non-mutagenic nitrated polycyclic aromatic hydrocarbons to mutagens, Mutation Res., 92, 29-37. Kasai, H., S. Nishimura, K. Wakabayashi, M. Nagao and T. Sugimura (1980) Chemical synthesis of 2-amino-3-methylimidazo-[4,3-f]quinoline (IQ), a potent mutagen isolated from broiled fish, Proc. Jpn. Acad., 56B, 382-384. Kawachi, T. (1982) Mutagenicity and carcinogenicity of nitropyrenes, in: D.E. Rickert (Ed.), The Toxicity of Nitroaromatic Compounds, Hemisphere, New York, in press. King, L.C., S.B. Tejada and J. Lewtas (1981) Evaluation of the release of mutagens and 1-nitropyrene from diesel particles in the presence of lung macrophage cells in culture, EPA Diesel Emission Symposium Abstract. Kohan, M., and L. Claxton (1981) Reduced bacterial mutagenicity for diesel exhaust extract and associated nitroarene compounds after metabolism and protein binding, EPA Diesel Emission Symposium Abstracts. Kriek, E. (1965) On the interaction of N-2-fluorenylhydroxylamine with nucleic acids in vitro, Biochem. Biophys. Res. Commun., 20, 793-799. Kriek, E., J.A. Miller, U. Juhl and E.C. Miller (1967) C8-(N-2-fluorenylacetamido)guanosine, an arylamidation reaction product of guanosine and the carcinogen N-acetoxy-N-2-fluorenylacetamide in neutral solution, Biochem., 6, 177-182. LaVoie, E.J., L. Tulley, V. Bedenko and D. Hoffmann (1981a) Mutagenicity of methylated fluorenes and benzofluorenes, Mutation Res., 91, 167-176. LaVoie, EJ., L. Tulley-Freiler, V. Bedenko and D. Hoffmann (1981b) Mutagenicity, tumor-intiating activity, and metabolism of methylphenanthrenes, Cancer Res., 41, 3441-3447. LaVoie, E.J., A. Goril, G. Briggs and D. Hoffmann (1981c) Mutagenicity of aminocarbazoles and nitrocarbazoles, Mutation Res., 90, 337-344. Lee, F.S.-C., T.M. Harvey, T.J. Prater, M.C. Paputa and D. Schuetzle (1980) Chemical analysis of diesel particulate matter and an evaluation of the artifact formation, in: Sampling and Analysis of Toxic Organics in the Atmosphere ASTM STP 721, American Society for Testing and Materials, pp. 92-110. Lefevre, J.-F., R.P.P. Fuchs and M.P. Daune (1978) Comparative studies on the 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene: Binding sites on DNA and conformation change of modified deoxytrinucleotides, Biochem., 17, 2561-2567. Levin, D.E., W.S. Barnes and E. Klekowski (1979) Mutagenicity of fluorene derivatives: A proposed mechanism, Mutation Res., 63, 1-10. Levine, A.F., L.M. Fink, I.B. Weinstein and D. Grunberger (1974) Effect of N-2-acetylfluorene modification on the conformation of nucleic acids, Cancer Res., 34, 319-327. Lewtas, J. (1982) Mutagenic activity of diesel emissions, in J. Lewtas (Ed.), Toxicological Effects of

261 Emissions from Diesel Engines, Elsevier Biomedical, Amsterdam, pp. 243-264. Lewtas, J., A. Austin and L. Claxton (1981) Diesel bus terminal study: Mutagenicity of the particle-bound organics and organic fractions, EPA Diesel Emissions Symposium Abstracts. Lewtas, J., A. Austin and L. Claxton (1982a) Characterization and comparison of the mutagenicity of respirable particles from urban air inside and outside a diesel bus terminal, Environ. Mutagen., 4, 408-409. Lewtas, J., M.G. Nishioka and B,A. Petersen (1982b) The role of nitroaromatics in the mutagenicity of environmental emissions, Abstracts, Fifth CIIT (Chemical Industry Institute of Toxicology) Conference on Toxicology: Toxicity of Nitroaromatic Compounds, p. 7. Li, A.P., C.R. Clark, R.H. Hanson, T.R. Henderson and C.H. Hobbs (1982) Coal-combustion fly ash extracts as direct-acting mutagen in Salmonella and promutagen in Chinese hamster ovary cells, Environ. Mutagen., 4, 407-408. Lindeke, B., E.G. Lundkvist, U. Jonsson and S.O. Eriksson (1975) Autoxidation of N-hydroxyamphetamine and N-hydroxyphentermine, Acta Pharm. Suecia, 12, 183. Lrfroth, G. (1981) Comparison of the mutagenic activity in carbon black particulate matter and in diesel and gasoline engine exhaust, in: M.D. Waters, S.S. Sandhu, J.L. Huisingh, L. Claxton and S. Nesnow (Eds.), Application of Short-Term Bioassays in the Analysis of Complex Environmental Mixtures, II. Environmental Science Research, Vol. 22, Plenum, New York, pp. 319-336. Lrfroth, G., E. Hefner, I. Alfheim and M. Moiler (1980) Mutagenic activity in photocopies, Science, 209, 1037-1039. LOfroth, G., R. Toftgard, J. Carlstedt-Duke, J.A. Gustafsson, E. Brorstrom, P. Grenafeld and A. Lindskog (1981) Effects of ozone and nitrogen dioxide present during sampling of genuine particulate matter as detected by two biological test systems and analysis of polycyclic aromatic hydrocarbons, EPA Diesel Emissions Symposium Abstract. Martin, C.N., A.C. McDermid and R.C. Garner (1978) Testing of known carcinogens and noncarcinogens for their ability to induce unscheduled DNA synthesis in HeLa cells, Cancer Res., 38, 2621-2627. McCalla, D.R., D. Voutsinos and P.L. Olive (1975) Mutagen screening with bacteria: Niridazole and nitrofurans, Mutation Res., 31, 31-37. McCann, J., N.E. Spingarn, J. Kobori and B.N. Ames (1975) Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids, Proc. Natl. Acad. Sci. (U.S.A.), 72, 979-983. McCarroU, N.E., B.H. Keech and C.E. Piper (1981a) A microsuspension adaptation of the Bacillus subtilis 'rec' assay, Environ. Mutagen., 3, 607-616. McCarroll, N.E., C.E. Piper and B.H. Keech (1981b) An E. coli microsuspension assay for the detection of DNA damage induced by direct-acting agents and promutagens, Environ. Mutagen., 3, 429-444. McCormick, J.J., and V.M. Maher (1981) Mutagenesis studies in diploid human cells with different DNA-repair capacities, in: H.F. Stich and R.H.C. Stan (Eds.), Short-Term Tests for Chemical Carcinogens, Springer, Heidelberg, pp. 264-276. McCoy, E.C., and H.S. Rosenkranz (1982) Cigarette smoking may yield nitroarenes, Cancer Lett., 15, 9-13. McCoy, E.C., L.A. Petrullo, H.S. Rosenkranz and R. Mermelstein (1981a) 4-Nitroquinoline-l-oxide: Factors determining its mutagenicity in bacteria, Mutation Res., 89, 151-159. McCoy, E.C., E.J. Rosenkranz, L.A. Petrullo, H.S. Rosenkranz and R. Mermelstein (1981b) Structural basis of mutagenicity in bacteria of nitrated naphthalene and derivatives, Environ. Mutagen., 3, 499-511. McCoy, E.C., E.J. Rosenkranz, L.A. Petrullo, H.S. Rosenkranz and R. Mermelstein (1981c) Frameshift mutations: Relative roles of simple intercalation and of adduct formation, Mutation Res., 90, 21-30. McCoy, E.C., E.J. Rosenkranz, H.S. Rosenkranz and R. Mermelstein (1981d) Nitrated fluorene derivatives are potent frameshift mutagens, Mutation Res., 90, 11-20. McCoy, E.C., H.S. Rosenkranz and R. Mermelstein (1981e) Evidence for the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens, Environ. Mutagen., 3, 421-427. McCoy, E.C., G.D. McCoy and H.S. Rosenkranz (1982) Esterification of arylhydroxylamines: Evidence for a specific gene product in mutagenesis, Biochem. Biophys. Res. Commun., 108, 1362-! 367.

262 McCoy, E.C., M. Anders, H.S. Rosenkranz and R. Mermelstein (1983) Apparent absence of recombinogenie activity of nitropyrenes for yeast, Mutation Res., 116, 119-127. Mermelstein, R., D.K. Kiriazides, M. Butler, E.C. McCoy and H.S. Rosenkranz (1981) The extraordinary mutagenicity of nitropyrenes in bacteria, Mutation Res., 89, 187-196. Mermelstein, R., E.C. McCoy and H.S. Rosenkranz (1982) The microbial mutagenicity of nitroarenes, in: R.R. Tice, D.L. Costa and K.M. Schaich (Eds.), The genotoxic Effects of Airborne Agents, Plenum, New York, pp. 369-396. Messier, F., C. Lu, P. Andrews, B.E. McCarry, M.A. Quilliam and D.R. McCalla (1981) Metabolism of l-nitropyrene and formation of DNA adducts in Salmonella typhimurium, Carcinogenesis, 2, 1007-1011. Monti-Bragadin, C., S. Venturini and P.A. Todd (1977) Interaction between two error-prone DNA repair systems in Escherichia coli, FEMS Microbiol. Lett., 2, 125-128. Moreau, P., A. Bailone and R. Devoret (1976) Prophage induction in Eseherichia coli K12 envA uvrB: A highly sensitive test for potential carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 73, 3700-3704. Moreau, P., and R. Devoret (1977) Potential carcinogens tested by inducation and mutagenesis of prophage ~ in Escheriehia co# KI2, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Book C, Cold Spring Harbor Laboratory, pp. 1451-1472. Mosberg, A., D. Mays, R. Riggin, M. Shure, J. Mumford and G. Fisher (1981) Chemical and physical properties of vapor phase nitropyrene-coated coal fly ash, in: Sixth International Symposium on Polynuclear Aromatic Hydrocarbons, Abstracts, Battelle Laboratories, Columbus, Ohio, p. 70. Mulvey, D., and W.A. Waters (1977) A mechanistic study of the decomposition of phenylhydroxylamine to azoxybenzene and aniline and its catalysis by iron (II) and iron (III) ions stabilized by ethylenediaminetetra-acetic acid, J. Chem. Soc., Perkin, II, 1868. Mumford, J.S., and J. Lewtas (1982) Sample collection and preparation factors which affect mutagenicity and cytotoxicity of coal fly ash, in: M.D. Waters et al. (Eds.), Third Symposium on the Application of Short-Term Bioassays in the Analysis of Complex Environmental Mixtures, Plenum, New York, in press. Nachtman, J.P., and S. Wolff (1982) Activity of nitro-polynuclear aromtic hydrocarbons in the sister chromatid exchange assay with and without metabolic activation, Environ. Mutagen., 4, 1-5. Nagao, M., T. Yahagi, T. Seino, T. Sugimura and H. Ito (1977) Mutagenicity of quinoline and its derivatives, Mutation Res., 42, 335-342. Nagao, M., K. Wakabayashi, H. Kasai, S. Nishimura and T. Sugimura (1981) Effect of methyl substitution on mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline, isolated from broiled sardine, Carcinogenesis, 2, 1147-1149. Nakayasu, M., H. Sakamoto, K. Wakabayashi, M. Terada, T. Sugimura and H.S. Rosenkranz (1982) Potent activity of nitropyrenes on Chinese hamster living cells with diphtheria toxin resistance as a selective marker, Carcinogenesis, 3, 917-922. National Academy of Sciences, U.S. (1981) Health effects of exposure to diesel exhaust, The Report of the Health Effects Panel of the Diesel Impact Study Committee, National Research Council--National Academy of Sciences, Washington, DC. Nestmann, E.R., E.G.-H. Lee, T.I. Matula, G.R. Douglas and J.C. Mueller (1980) Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/mammalian-microsome assay, Mutation Res., 79, 203-212. Nilsson, L., G. LOfroth, R. Toftgard and T. Greibrokk (1981) Nitroarenes: Mutagenicity in the Ames Salmonella/microsome assay and affinity to the TCDD-receptor protein, Eur. Environ. Mutagen. Soc. (Abstract). Nishioka, M.G., B.A. Petersen and J. Lewtas (1981) Comparison of nitro-PNA content and mutagenicity of diesel emissions, EPA Diesel Emission Symposium Abstract. Oglesby, L.A., T.J. Flammang, D.L. Tullis and F.F. Kadlubar (1981) Rapid absorption, distribution, and excretion of carcinogenic N-hydroxy-arylamines after direct urethral instillation into the rat urinary bladder, Carcinogenesis, 2, 15-20. Ohgaki, H., N. Matsukara, K. Morino, T. Kawachi, T. Sugimura, K. Morita, H. Tokiwa and T. Hirota (1982) Carcinogenicity in rats of the mutagenic compounds l-nitropyrene and 3-nitrofluoranthene, Cancer Lett., 15, 1-7.

263 Ohnishi, Y., T. Kinouchi and K. Wakisaka (1981) Decrease of mutagenic activity of l-nitropyrenes in intestinal bacteria, Third International Conference on Environmental Mutagens, Abstracts, p. 84. Ohnishi, Y., T. Kinouchi, Y. Manabe and K. Wakisaka (1982) Environmental aromatic nitro compounds and their bacterial detoxification, in: M.D. Waters et al. (Eds.), Third Symposium on Application of Short-Term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures, Plenum, New York, in press. Oldham, J., F.F. Kadlubar and G.E. Milo (1981) Effect of pH on the neoplastic transformation of normal human skin fibroblasts by N-hydroxyl-l-naphthylamine and N-hydroxy-2-naphthylamine, Carcinogenesis, 2, 937-940. Painter, R.B. (1981) DNA synthesis inhibition in mammalian cells as a test for mutagenic carcinogens, in H.F. Stich and R.H.C. San (Eds.), Short-Term Tests for Chemical Carcinogens, Springer, Heidelberg, pp. 59-64. Painter, R.B., and R. Howard (1982) The HeLa DNA synthesis inhibition test as a rapid screen for mutagenic carcinogens, Mutation Res., 92, 427-437. Pederson, T.C., and J.-S. Siak (1981a) The role of nitroaromatic compounds in the direct-acting mutagenicity of diesel particle extracts, J. Appl. Toxicol. 1, 54-60. Pederson, T.C., and J.-S. Siak (1981b) The activation of mutagens in diesel particle extract with rat liver $9 enzymes, J. Appl. Toxicol. 1, 61-66. Pederson, T.C., and J.-S. Siak (1981c) Dinitropyrenes: Their probable presence in diesel particle extracts and consequent effect on mutagenic activations by NADH-dependent $9 enzymes, EPA Diesel Emissions Symposium Abstract. Pederson, T.C., and J.-S. Siak (1982a) The cooperative role of bacterial and mammalian enzymes in mutagenic activation of nitro-PAH compounds, Abstracts, Fifth CIIT (Chemical Industry Institute of Toxicology) Conference on Toxicology: Toxicity of Nitroaromatic Compounds, p. 3. Pederson, T.C., and J.-S. Siak (1982b) Mutagenic activation of nitro-PAH by microsomal and cytosolic enzymes, Soc. Toxicol., Abstract. Ann. Mutagen., p. 49. Petersen, B.A., C.C. Chuang, W.L. and Margard and D.A. Trayser (1981) Identification of mutagenic compounds in extracts of diesel exhaust particulates, Air Poll. Control Assoc. Abstract, 74th Annual Meeting, 81-56.1. Pienta, R.J. (1980) Evaluation and relevance of the Syrian hamster embryo cell system, in: G.M. Williams, R. Kroes, H.W. Waaijers and K.W. Van de Poll, (Eds.), The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier/North-Holland, Amsterdam, pp. 149-169. Pitts, J.N., Jr. (1979) Photochemical and biological implications of the atmospheric reactions of amines and benzo[a]pyrene, Phil. Trans. Roy. Soc. London, Ser. A, 290, 551-576. Pitts, J.N., Jr., K.A. van Cauwenberghe, D. Grosjean, J.P. Schmid, D.R. Fitz, W.L. Belser Jr., G.B. Knudson and P.M. Hunds (1979) Atmospheric reactions of polycyclic aromatic hydrocarbons: facile formation of mutagenic nitro derivatives, Science, 202, 515-519. Pitts, J.N., Jr., W. Harger, D.M. Lokensgard, D.R. Fitz, G.M. Scorziell and V. Mgjia (1982a) Diurnal variations in the mutagenicity of airborne particulate organic matter in California's south coast air basin, Mutation Res., 104, 35-41. Pitts, J.N., Jr., D.M. Lokengard, W. Harger, T.S. Fisher, V. Majia, J.J. Sehuler, G.M. Scorziell and Y.A. Katzenstein (1982b) Mutagens in diesel exchaust particulate: Identification and direct activities of 6-nitrobenzo[a]pyrene, 9-nitroanthracene, 1-nitropyrene and 5H-phenanthro[4,5-bcd]pyran-5-one, Mutation Res., 103, 241-249. Poirier, L.A., and J.H. Weisburger (1974) Enzymic reduction of carcinogenic aromatic nitro compounds by rat and mouse liver fractions, Biochem. Pharmacol., 23, 661-669. Poirier, L.A., and E.K. Weisburger (1979) Selection of carcinogens and related compounds tested for mutagenic activity, J. Natl. Cancer Inst., 62, 833-840. Prater, T.J., T. Riley and D. Schuetzle (1981) Capillary column G C / M S characterization of diesel exhaust particulate extracts, EPA Diesel Emissions Symposium Abstracts. Pueyo, C. (1978) Forward mutations to arabinose resistance in Salmonella typhimurium strains, Mutation Res., 54, 311-321. Purchase, I.F.H., E. Longstaff, J. Ashby, J.A. Styles, D. Anderson, P.A. Lefevre and F.R. Westwood

264 (1978) An evaluation of six short4erm tests for detecting organic chemical carcinogens, Br. J. Cancer, 37, 873-959. Quilliam, M.A., F. Messier, C. Lu, P.A. Andrews, B.E. McCarry and D.R. McCalla (1982) The metabolism of nitro-substituted polycyclic aromatic hydrocarbons in Salmonella typhimurium, in: Proceedings of the International Conference on Polycyclic Aromatic Hydrocarbons, Vol. 5, Battelle Laboratories, Columbus, Ohio, in press. Radman, M. (1977) Inducible pathways in deoxyribonucleic acid repair, mutagenesis and carcinogenesis, Biochem. Soc. Trans., 5, 1194-1199. Radman, M. (1980) Is there SOS induction in mammalian cells, Photochem. Photobiol., 32, 823-830. Radman, M., G. Villani, S. Boiteux, M. Defais, P. Caillet-Fauquet and S. Spadari (1977) On the mechanism and genetic control of mutagenesis induced by carcinogenic mutagens, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Book B, Cold Spring Harbor Laboratory, Cold Spring Harbor, pp. 903-922. Radomski, J.L., G.M. Conzelman Jr., A.A. Rey and E. Brill (1973) N-Oxidation of certain aromatic amines, acetamides and nitro compounds by monkey and dogs, J. Natl. Cancer Inst., 50, 989-995. Rappaport, S.M., Y.Y. Wang, E.T. Wei, R. Sawyer, B.E. Watkins and H. Rapoport (1980) Isolation and identification of a direct-acting mutagen in diesel-exhaust particulates, Environ. Sci. Tech., 14, 1505-1509. Riley, T., T. Prater, D. Schuetzle, T.M. Harvey and D. Hunt (1981) The analysis of nitrated polynuclear aromatic hydrocarbons in diesel exhaust particulates by mass spectrometry/mass spectrometry techniques, EPA Diesel Emission Symposium Abstract. Rosenkranz, H.S. (1982) Direct-acting mutagens in diesel exhausts: Magnitude of the problem, Mutation Res., 101, 1-10. Rosenkranz, H.S., and Z. Leifer (1980) Detection of carcinogens and mutagens with DNA repair-deficient bacteria, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Vol. 6, Plenum, New York, pp. 109-147. Rosenkranz, H.S., and R. Mermelstein (1980) The Salmonella mutagenicity and the E. coli Pol A+/Pol A~- repair assays: Evaluation of relevance to carcinogenesis, in: G.M. Williams, R. Kroes, H.W. Waaijers and K.W. van de Poll (Eds.), The Predictive Value of Short-Term Screening Tests in the Evaluation of Carcinogenicity, Elsevier/North-Holland, Amsterdam, pp. 5-26. Rosenkranz, H.S., and L.A. Poirier (1979) An evaluation of the mutagenicity 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., and W.T. Speck (1976) Activation of nitrofurantoin to a mutagen by rat liver nitroreductase, Biochem. Pharmacol., 25, 1555-1556. Rosenkranz, H.S., G. Karpinsky and E.C. McCoy (1980a) Microbial assays: Evaluation and application to the elucidation of the etiology of cancer, in: K. Norpoth and R.C. Garner (Eds.), Short-Term Mutagenicity Test Systems for Detecting Carcinogens, Springer, Berlin, pp. 19-57. Rosenkranz, H.S., E.C. McCoy, D.R. Sanders, M. Butler, D.K. Kiriazides and R. Mermelstein (1980b) Nitropyrenes: isolation, identification and reduction of mutagenic impurities in carbon black and toners, Science, 209, 1039-1043. 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 detection of nitroarenes as mutagens in complex mixtures including diesel exhausts, Mutation Res., 91, 103-105. Rosenkranz, E.J., E.C. McCoy, R. Mermelstein and H.S. Rosenkranz (1982a) Evidence for the existence of distinct nitroreductases in Salmonella typhimurium: Roles in mutagenesis, Carcinogenesis, 3, 121-123. Rosenkranz, H.S., G.E. Karpinsky, M. Anders, E.J. Rosenkranz, L.A. Petrullo, E.C. McCoy and R. Mermelstein (1982b) Adaptability of microbial mutagenicity assays to the study of problems of environmental concern, in: C.W. Lawrence, L. Prakash and F. Sherman (Eds.), Induced Mutagenesis: Molecular Mechanisms and their Implications for Environmental Protection, Plenum, New York. Rosenkranz, H.S., E.C. McCoy and R. Mermetstein (1982c) Microbial assays in research and in the

265 characterization of complex mixtures, in: M.D. Waters et al. (Eds.), Third Symposium on Application of Short-Term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures, Plenum, New York, in press. Rosenkranz, H.S., E.C. McCoy and R. Mermelstein (1982d) Nitrated polycyclic aromatic hydrocarbons: A newly recognized group of ubiquitous environmental agents, in: I.B. Weinstein and H.J. Vogel (Eds.), Genes and Proteins in Oncogenesis, Academic Press, New York, in press. Sage, E., and M. Leng (1980) Conformation of poly(dG-dC).poly(dG-dC) modified by the carcinogens N-acetoxy-N-acetyl-2-aminofluorene and N-hydroxy-N-2-aminofluorene, Proc. Natl. Acad. Sci. (U.S.A.) 77, 4597-4601. Sage, E., R.P.P. Fuchs and M. Leng (1979) Reactivity of the antibodies to DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene, Biochem., 18, 1328-1332. Saimeen, I., A.M. Durisin, T.J. Prater, T. Riley and D. Schuetzle (1982) Contribution of l-nitropyrene to direct-acting Ames assay mutagenicities of diesel particulate extracts, Mutation Res., 104; 17-23. Santella, R.M., R.P.P. Fuchs and D. Grunberger (1979) Mutagenicity of 7-iodo and 7-fluoro derivatives of N-hydroxy- and N-acetoxy-N-2-acetylaminofluorene in the Salmonella typhimurium assay, Mutation Res., 67, 85-87. Sargentini, N.J., and K.C. Smith (1981) Much of spontaneous mutagenesis in Escherichi coli is due to error-prone DNA repair: Implications for spontaneous carcinogenesis, Carcinogenesis, 2, 863-872. Schuetzle, D., F.S.C. Lee, T.J. Prater and S.B. Tejada (1981) The identification of polynuclear aromatic hydrocarbon (PAH) derivatives in mutagenic fractions of diesel particulate extracts, Intern. J. Environ. Anal. Chem., 9, 1-52. Schuetzle, D., T.L. Riley, T.J. Prater, T.M. Harvey and D.F. Hunt (1982a) Analysis of nitrated polycyclic aromatic hydrocarbons in diesel particulates, Anal. Chem., 54, 265-271. Schuetzle, D., T.L. Riley, T.J. Prater and I. Salmeen (1982b) The identification of mutagenic chemical species in air particulate samples, in: J. Albaiges (Ed.), Analytical Techniques in Environmental Chemistry, II, Pergamon, Oxford, pp. 259-280. Scribner, J.D., S.R. Fisk and N.K. Scribner (1979) Mechanisms of action of carcinogenic aromatic amines: An investigation using mutagenesis in bacteria, Chem.-Biol. Interact., 26, 11-25. Simmon, V.F. (1979a) In vitro mutagenicity assays of chemical carcinogens and related compounds with Salmonella typhimurium, J. Natl. Cancer Inst., 62, 893-899. Simmon, V.F. (1979b) In vitro assays for recombinogenic activity of chemical carcinogens and related compounds with Saccharomyces cerevisiae D3, J. Natl. Cancer Inst., 62, 901-909. Simmon, V.F., H.S. Rosenkranz, E. Zeiger and L.A. Poirier (1979) Mutagenic activity of chemical carcinogens and related compounds in the intraperitoneal host-mediated assay, J. Natl. Cancer Inst., 62, 911-918. Sivak, A., M.C. Charest, L. Rudenko, D.M. Silveira, I. Simons and A.M. Wood (1981) Balb/c-3T3 cells as target cells for chemically induced neoplastic transformation, in: N. Mishra, V. Dunkel and M. Mehlman (Eds.), Mammalian Cell Transformation by Chemical Carcinogens, Senate, Princeton Junction, NJ;pp. 133- i 80. Speck, W.T., and H.S. Rosenkranz (1980) Proflavin: An unusual mutagen, Mutation Res., 77, 37-43. Spodheim-Maurizot, M., G. Saint-Ruf and M. Leng (1980) Antibodies to N-hydroxy-2-aminofluorene modified DNA as probes in the study of DNA reacted with derivatives of 2-acetylaminofluorene, Carcinogen., 1,807-812. Styles, J.A. (1981) Use of BHK 21/C1 13 cells for chemical screening, in: N. Mishra, V. Dunkel and M. Mehlman (Eds.), Mammalian Cell Transformation by Chemical Carcinogens, Senate, Princeton Junction, NJ, pp. 85-131. Suter, W., and I. Jaeger (1982) Comparative evaluation of different bacterial DNA repair tests for rapid detection of chemical mutagens and carcinogens, Mutation Res., 97, 1-18. Talcott, R.E., and W. Harger (1981) Chemical characterization of direct-acting airborne mutagens: The functional group, Mutation Res., 91,433-436. Tazima, Y., T. Kada and A. Murakami (1975) Mutagenicity of nitrofuran derivatives including furylfuramide, a food preservative, Mutation Res., 32, 55-80. Todd, P.A., C. Monti-Bragadin and B.W. Glickman (1979) MMS mutagenesis in strains of Escherichia

266 coli carrying the R46 mutagenic enhancing plasmid: Phenotypic analysis of Arg + revertants, Mutation Res., 62, 227-237. Tokiwa, H., R. Nakagawa, K. Morita and Y. Ohnishi (1981a) Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to nitrogen dioxide, Mutation Res., 85, 195-205. Tokiwa, H., R. Nakagawa and Y. Ohnishi (1981b) Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to gaseous pollutants, Third International Conference on Environmental Mutagens, Abstracts, p. 79. Tokiwa, H., R. Nakagawa and Y. Ohnishi (1981c) Mutagenic assay of aromatic nitro compounds with Salmonella typhimurium, Mutation Res., 91,321-325. Tong, S., and J.K. Selkirk (1982) Metabolism of 6-nitrobenzo[a]pyrene by hamster embryo cells and rat liver microsomes, Prec. Am. Assoc. Cancer Res., 23, 80. Topham, J.C. (1980) Do induced sperm-head abnormalities in mice specifically identify mammalian mutagens rather than carcinogens? Mutation Res., 74, 379-387. Troll, W., S. Belman and E. Levin (1963) The effect of metabolites of 2-naphthylamine and the mutagen hydroxylamine on the thermal stability of DNA and polyribonucleotides, Cancer Res., 23, 841-847. Tu, A.S., A. Sivak and R. Mermelstein (1982) Evaluation of in vitro transforming ability of nitropyrenes, Abstracts Fifth CIIT (Chemical Industry Institute of Toxicology) Conference on Toxicology: Toxicity of Nitroaromatic Compounds. U.S. Environmental Protection Agency (1978) Health effects associated with diesel exhaust emissions, EPA-600/1-78-063. Walker, G.C. (1978) Isolation and characterization of mutants of the plasmid pKMI01 deficient in their ability to enhance mutagenesis and repair, J. Bacteriol., 133, 1203-1211. Wang, C.Y., K. Muraoka and G.T. Bryan (1975) Mutagenicity of nitrofurans, nitrothiophenes, nitropyrroles, nitroimidazole, aminothiophenes and aminothiazoles in Salmonella typhimurium, Cancer Res., 36, 3611-3617. Wang, Y.Y., S.M. Rappaport, R.F. Sawyer, R.E. Talcott and E.T. Wei (1978) Direct-acting mutagens in automobile exhaust, Cancer Lett., 5, 39-47. Wang, C.H., M.-S. Lee, C.M. King and P.O. Warner (1980) Evidence for nitroaromatics as direct-acting mutagens of airborne particulates, Chemosphere, 9, 83-87. Waring, M.J., A. Gonzalez, A. Jimenez and D. Vazquez (1979) Intercalative binding to DNA of antitumour drugs derived from 3-nitro-l,8-naphthalic acid, Nucleic Acids Res., 7, 217-230. Wei, C.I., O.G. Raabe and L.S. Rosenblatt (1982) Microbial detection of mutagenic nitro-organic compounds in filtrates of coal fly ash, Environ. Mutagen., 4, 382. Weill-Thevenet, N., J.-P. Buisson, R. Royer and M. Hofnung (1981) Mutagenic activity of benzofurans and naphthofurans in the Salmonella/microsome assay: 2-Nitro-7-methoxynaphtho[2,l-b]furan (R7000), a new highly potent mutagenic agent, Mutation Res., 88, 355-362. Weisburger, E.K., and J.H. Weisburger (1958) Chemistry, carcinogenicity, and metabolism of 2-fluorenamine and related compounds, Adv. Cancer Res., 5, 331-431. Weisburger, J.H., and E.K. Weisburger (1973) Biochemical formation and pharmacological, toxicological and pathological properties of hydroxylamines and hydroxamic acids, Pharm. Rev., 25, 1-66. Westra, J.G., and A. Visser (1979) Quantitative analysis of N-(guanin-8-yl)-N-acetyl-2-aminofluorene and N-(guanin-8-yl)-2-aminofluorene in modified DNA by hydrolysis in trifluoroacetic acid and high pressure liquid chromatography, Cancer Lett., 8, 155-162. Westra, J.G., E. Kriek and H. Hittenhausen (1976) Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo. Chem.-Biol. Interact., 15, 149-164. Wilcox, P., and J.M. Parry (1981) The genetic activity of dinitropyrenes in yeast: Unusual dose-response curves for induced mitotic gene conversion, Carcinogenesis, 2, 1201-1206. Wilcox, P., N. Danford and J.M. Parry (1982) The genetic activity and metabolism of dinitropyrenes in eucaryotic cells, in: Progress in Genetic Toxicology, Elsevier/North-Holland, Amsterdam, in press. Wirth, P.J., P. Alewood, I. Calder and S.S. Thorgeirsson (1982) Mutagenicity of N-hydroxy-2acetylaminofluorene and N-hydroxyphenacetin and their respective deacetylated metabolites in nitroreductase-deficient Salmonella TA98FR and TA 100FR, Carcinogenesis, 3, 167-170. Wong, T.K., P.-L. Chiu, P.P. Fu and S.K. Yang (1981) Metabolic study of 7-methylbenzo[a]pyrene with

267 rat liver microsomes: Separation by reversed-phase and normal-phase high performance liquid chromatography and characterization of metabolites, Chem.-Biol. Interact., 36, 153-166. Wyrobeck, A.J., and W.R. Bruce (1978) The induction of sperm-shape abnormalities in mice and human, in: A. HoUaender and F.J. de Serres (Eds.), Chemical Mutagens, Vol. 5, Plenum, New York, pp. 257-285. Xu, X.B., J.P. Nachtman, Z.L. Jin, E.T. Wei, S. Rappaport and A.L. Burlingame (1981) Isolation and identification of mutagenic nitroarenes in diesel exhaust particulates, EPA Diesel Emissions Symposium Abstract. Xu, X.B., J.P. Nachtman, S.M. Rappaport, E.T. Wei, S. Lewis and A.L. Burlingame (198 l a) Identification of 2-nitrofluorene in diesel exhaust particulates, J. Appl, Toxicol., 1, 196-198. Xu, X.B., J.P. Nachtman, Z.L. Jin, E.T. Wei and S.M. Rappaport (1982) Isolation and identification of mutagenic nitro-PAH in diesel-exhaust particulates, Anal. Chim. Acta, 136, 163-174. Yahagi, T., M. Nagao, K. Hara, T. Matsushima, T. Sugimura and G.T. Bryan (1974) Relationships between the carcinogenic and mutagenic or DNA-modifying effects of nitrofuran derivatives, including 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, a food additive, Cancer Res., 34, 2266-2273. Yahagi, T., H. Shimizu, M. Nagao, N. Takemura and T. Sugimura (1975) Mutagenicity of 5nitroacenaphthene in Salmonella, Gann, 66, 581-582. Yamasaki, H., P. Pulkrabek, D. Grunberger and I.B. Weinstein (1977) Differential excision from DNA of the C-8 and N z guanosine adducts of N-acetyl-2-aminofluorene by single strand-specific endonucleases, Cancer Res., 37, 3756-3760. Yergey, J.A., T.H. Risby and S.S. Lestz (1981) The chemical characterization of diesel particulate matter, EPA Diesel Emissions Symposium Abstract. Yoshikura, H., T. Kuchino and T. Matsushimu (1979) Carcinogenic chemicals enhance mouse leukemia virus infection in contact-inhibited culture: A new simple method of screening carcinogens, Cancer Lett., 7, 203-208. Ziegler, D.M., E.M. McKee and L.L. Poulsen (1973) Microsomal flavoprotein-catalyzed N-oxidation of arylamines, Drug Metabolism and Disposition, 1,314-321. Zweidinger, R.B. (1981) Emission factors from diesel and gasoline powered vehicles; Correlation with the Ames test, EPA Diesel Emission Symposium Abstract.