Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls

Mutation Research, 163 (1986) 101-107

101

Elsevier MTR 04244

Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls Teruhisa Hirayama ~, Haruo Kusakabe, Tetsushi Watanabe, Shigeru Ozasa, Yasuhiro Fujioka and Shozo Fukui Kyoto Pharmaceutical University, 5-Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607 (Japan) (Received 5 March 1986) (Revision received 26 May 1986) (Accepted 28 May 1986)

Summary Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP 6 in the absence of $9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP 6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,SDNP 6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP 6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.

A number of nitroaromatic compounds have been used as industrial chemical materials (Howard et al., 1976), and are of interest because of their mutagenicity in bacterial tester strains and carcinogenicity in experimental animals (McCann et al., 1975; Rinkus and Legator, 1979; Rosenkranz and Mermelstein, 1983). Recently, increasing attention has been given to the presence of potent mutagenic nitroarenes in environmental samples, including airborne particulates, photocopy toners and diesel particulates (Rosenkranz et al., 1980; Pitts et al., 1982; Xu et al., 1982; Handa et al., 1983; Hadson et al., 1983; Pitts, 1983; Tokiwa et al., 1983; Yu et al., 1984). Nitroaromatic * Author to whom correspondence should be addressed.

compounds are reduced enzymatically to arylhydroxylamines which may be converted to the putative ultimate mutagens, the arylnitrenium ion, an adduct to the nucleophile site of DNA bases. Several studies on the relationships between chemical structures of nitroaromatics and their mutagenic activities have been reported (Mermelstein et al., 1984; Greibrokk et al., 1984; Lofroth et al., 1984; Rosenkranz et al., 1984; Vance and Levin, 1984; Vance et al., 1985). Biphenyl is a common component of organic environmental pollutants and has been detected in air (Karasek et al., 1978), synthetic fuels derived from oil shale, tar sands and coal (Later et al., 1981), and combustion products of coal and wood (Lee et al., 1977).

0027-5107/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

102 It may, therefore, be converted into mutagenic nitro derivatives in an atmosphere containing nitrogen oxide, and 4-nitrobiphenyl (Deichmann et al., 1958) and 4,4'-dinitrobiphenyl (Laham et al., 1964) are well known carcinogens. A previous paper (Nohara et al., 1985) described the relationship between the chemical structure of amino- and nitrobiphenyls and their mutagenic potency in Salmonella typhimurium strains. In the case of nitrobiphenyl, the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2-position. In the present study, we describe the relationship between the mutagenic potency and chemical structure of nitro-substituted biphenyl. We consider the number of substituents (mono-, di-, triand tetra-) and the position of substituents (ortho, meta and para). Three Salmonella typhimurium strains (TA98, TA98NR and TA98/1,8DNP6) were used in the absence of a mammalian metabolic activation system, in order to elucidate the mechanism of activation of the nitrobiphenyls. Materials and methods

Chemicals 2,4,3'- and 3,4,4'-trinitrobiphenyls were synthesized from 3,4'-dinitrobiphenyl, and 3,4,3'-trinitrobiphenyl and 3,4,3',4'-tetranitrobiphenyl were synthesized from 3,3'-dinitrobiphenyl by the method of Case (1942). 2,4,4'- and 2,4,2'-trinitrobiphenyls were synthesized from 4,4'- and 2,2'-dinitrobiphenyls, respectively, and 2,4,2',4'-tetranitrobiphenyl was obtained from further nitration of 2,4,4'-trinitrobiphenyl with sulfuric acid and nitric acid as described by Gull and Turner (1929). 2A,Y,4'-Tetranitrobiphenyl was synthesized from 2,3'-dinitrobiphenyl as described by Blakey and Scarborough (1927). 2,4- and 3,5-dinitrobiphenyls were obtained from the modified method of the Gomberg, Bachman and Hey (Elks et al., 1940) reaction of 2,4- and 3,5-dinitroaniline, respectively, with benzene. 2,6,2'- (m.p. 135.5°C), 2,6,3'(m.p. 161.5°C), 2,6,4'-trinitrobiphenyl (m.p. 188.2°C) and 2,4,2',6'-tetranitrobiphenyl (m.p. 227.0°C) were obtained from the Ullmann reaction of 2,6-dinitro-l-chlorobenzene with o-, m-, p-iodonitrobenzene and 2,4-dinitro-l-chloroben-

zene, respectively, and 2,6,2',6'-tetranitrobiphenyl (m.p. 219.4°C) was obtained from the above reaction of 2,6-dinitro-l-chlorobenzene alone (Wirth et al., 1964). Mono- and dinitrobiphenyl were synthesized as described by Nohara et al. (1985). Chemical purities were confirmed by melting point measurement, elemental analysis, and gas chromatography. The chemicals, whose purities are above 99%, were used for the mutagenicity test. 4-Nitroquinoline N-oxide (4NQO) was purchased from Nakarai Chemicals Ltd., Kyoto, Japan. Dimethyl sulfoxide (DMSO) (spectrophotometric grade) was obtained from E. Merck, Darmstadt, F.R.G.

Mutagenicity test The mutagenicity tests were performed essentially by the Ames method (Ames et al., 1975; Maron and Ames, 1983) with the suspension assay modification of Yahagi (1975). The assays were carried out in vitro on 3 histidine-requiring strains of Salmonella typhimurium (TA98, TA98NR and TA98/1,SDNP6). Each tester strain was routinely checked, without metabolic activation, for optimal response to a known mutagenic chemical, 4NQO (0.5 #g/plate). Every chemical was tested on at least 3 separate occasions and every dose level was assayed in triplicate. Mutagenic activity was calculated from the linear (ascending) portion of the dose-response curve. Mutability was exhibited as revertants/nmole sample. Results and discussion

The maximum concentration of the compound tested was settled at 1000 /Lg/ml in DMSO (100 /~g/plate), because the solution of more than 1000 /~g/ml of nitrobiphenyls brought about precipitation of the samples at the preincubation step. The mutagenic activities in Salmonella typhimurium TA98, TA98NR and T A 9 8 / 1 , 8 D N P 6 of all positional isomers of mononitrobiphenyl, and those of di-, tri- and tetranitrobiphenyls are given in Table 1. TA98NR is the so-called "classical" nitroreductase-deficient strain (Rosenkranz and Speck, 1975; Rosenkranz and Poirier, 1979; Rosenkranz and Mermelstein, 1980). This strain lacks a nitroreductase required for the activation of nitrofuran, nitroimidazole, nitrofluorenes and

103

other nitroarenes to mutagens (Quilliam et al., 1982; Rosenkranz and Poirier, 1979; Rosenkranz and Mermelstein, 1980). Strain TA98/1,8DNP 6 was isolated as resistant to the mutagenicity of 1,8-dinitropyrene (McCoy et al., 1981). This strain is also resistant to the mutagenicity of nitrofluorenes, other nitroarenes and N-hydroxy2-acetylaminofluorene (McCoy et al., 1983), and lacks esterification enzymes (McCoy et al., 1983). 3 of the mononitro-, 8 of the dinitro- and 5 of the trinitrobiphenyl derivatives were contained in group 1. In the mutagenicity of mononitrobiphenyls and dinitrobiphenyls with one substituent on each benzene ring, the structural requirements favoring mutagenic activity are the

TABLE

1

MUTAGENICITY SENCE

presence of a nitro group at the 4-position and its absence at the 2-position as described previously (Nohara et al., 1985). Of 2 dinitrobiphenyls (2,4and 3,5-dinitrobiphenyl) and 5 trinitrobiphenyls tested, two were mutagenic. The criterion for mutagenicity was 100 ~tg/plate, this being more than 3-fold that of the solvent controls. Of the two mutagenic compounds, 2,4,3'-trinitrobiphenyl was mutagenic in TA98, whereas 2,4,4'-trinitrobiphenyl was mutagenic in strains TA98 and TA98/ 1,8DNP6 on the above criteria. In group 1, 4,4'-dinitrobiphenyl and 2,4,4'-trinitrobiphenyl have potency in TA98 and TA98/1,SDNP 6. In group 2, these compounds possessed the adjoining dinitro group (3,4-dinitro group), and all

OF

NITROBIPHENYLS

IN

Salmonella t)Thimurium T A 9 8 ,

TA98NR

AND

TA98/1,8DNP~

IN THE

AB-

OF $9 (net revertants/nmole)

Group

Substitution

1

2-NBP

TA98 -

TA98NR

TA98/1,8DNP

2,2'-DNBP

-

-

-

3-NBP

-

-

-

3,5-DNBP

-

-

-

3,3'-DNBP

-

-

-

6

-

2,3'-DNBP

2

3

4-NBP

0.51

-

0.43

4,2'-DNBP

-

-

-

4,3'-DNBP

1.68

-

4,4'-DNBP

14.78

-

2,4-DNBP

-

2,4,6-TNBP

-

1.07

-

4.53

-

1,95

2,6,3'-TN BP

-

-

-

2,6,4'-TNBP

-

3,4,3'-TNBP

83.35

11.76

47.72

3,4,4'-TNBP

400.29

87.28

188.44

2,4,3',4'-TNBP*

33.44

3.44

8.12

3,4,Y,4'-TNBP*

715.72

144.82

273.24

2,4,2'-TNBP

0.64

-

-

2,6,2'-TN BP

-

-

-

2,6,2',6'-TN BP*

-

-

-

2,4,2',6 '-TN BP *

0.86 457.19

Benzo[ e ]cinnoline-6-oxide

DNBP,

-

2,4,4'-TNBP

267.56

-

2-Nitrobenzo[ c ]cinnoline-6-oxide

NBP, nitrobiphenyl;

-

2,4,3'-TNBP

2,4,2',4'-TNBP*

-, data given are not linear dose-response control.

10.93

NT

139.06 curves and the number

dinitrobiphenyl;

TNBP,

49,16

of revertants/100

trinitrobiphenyl;

TNBP*,

298.66

NT

# g s a m p l e is less t h a n 3 - f o l d t h a t o f s o l v e n t

tetranitrobiphenyl.

NT, not tested.

104

of these compounds have direct-acting mutagenicity (i.e. not requiring $9 enzymes, but still requiring bacterial enzymes) in TA98 and T A 9 8 / 1,8DNP 6, and retain the activity in TA98NR, as if they did not require classical nitroreductase to obtain their mutagenicity. Introduction of a nitro group at the 4'-position of 3,4-dinitrobiphenyl (3,4,4'-trinitrobiphenyl) formed a more mutagenic compound than that with a nitro group at the 3'-position (3,4,3'-trinitrobiphenyl). Furthermore, the most potent mutagen was obtained by introducing a nitro group at the 3'-position instead of the 2'-position of 3,4,4'-trinitrobiphenyl, The structural requirements of this group favoring mutagenic activity are not only the presence of an adjoining dinitro group but also the presence of a nitro group at the 4-position and its absence at the 2-position, the same as in the case of group 1. However, the role of the nitro group at the 3-position of nitrobiphenyls on their mutability in Salmonella typhimurium strains was not solved clearly. In group 3, these compounds were derivatives of 2,2'-dinitrobiphenyl, which may not have mutagenie potency in Salmonella typhimurium strains. Although, out of 2 trinitro- and 3 tetranitrobiphenyls tested, 3 compounds were mutagenic. Of 3 mutagenic compounds, one (2,4,2',4'-tetranitrobiphenyl) was mutagenic in 3 strains, TA98, TA98NR and T A 9 8 / 1 , 8 D N P 6, while two (2,4,2'trinitrobiphenyl and 2,4,2',6'-tetranitrobiphenyl) were mutagenic only in TA98 on the above criteria. 2,4,2',4'-Tetranitrobiphenyl was the most potent mutagen in TA98NR in the present study. In addition, we investigated the spectrophotometric and polarographic studies on nitrobiphenyls for the explanation of their mutability in the TA98 series. Data for the maxima of K-bands of biphenyl, o-, m- and p-nitrobiphenyl, and their p-nitro derivatives in cyclohexane are shown in Table 2, together with the displacement of )~ma~lnm) due to the introduction of the nitro group. )~,n~,, at 247 nm of biphenyl was shifted to a longer wavelength at 295 nm (bathochromic shift) by the introduction of the nitro group (4-nitrobiphenyl). The introduction of the p-nitro group into 2- and 3-nitrobiphenyl, to give 2,4'- and 3,4'-dinitrobiphenyl respectively, has a similar effect on their

TABLE 2 RED SHIFT OF )~nl~,x DUE TO THE NITRO SUBSTITUTION ON 4-POSITION OF BIPHENYLS Parent

4-Substituted

)'m~x shift

Compounds

X,,.... (nm)

Compounds

)~max (nm)

(nm)

Biphenyl (BP) 2-NBP 3-NBP 4-NBP

247 232 246 295

4-NBP 2,4'-DNBP 3,4'-DNBP 4,4'-DNBP

295 261 276 295

+48 +29 + 30 0

spectra. In the case of 4-nitrobiphenyl, ~,n~ at 295 nm was shifted to neither longer nor shorter wavelength by the introduction of a p-nitro group. Berry et al. (1960) reported that the negligible band displacement ( - 0 . 8 nm), to shorter wavelength, is observed on the introduction of the second p-nitro group to biphenyl, but intensity (Zm~) of 4,4'-dinitrobiphenyl increased 1.5 times that of 4-nitrobiphenyl. Data for the maxima of K-bands of biphenyl, o-, m- and p-nitrobiphenyl, 2,4'-, 3,4'- and 4,4'-dinitrobiphenyl, and their 2'-nitro derivatives in cyclohexane are shown in Table 3, together with the displacement of )'ma~¢nm~due to the introduction of the nitro group at the 2'-position. As can be seen in Table 3, biphenyl, 4-nitrobiphenyl, 2,4'-, 3,4'- and 4,4'-dinitrobiphenyl were shifted to shorter wavelength (hypsochromic shift) by the introduction of the 2'-nitro substituent. In the case of 3-nitrobiphenyl, a certain Xn,a~ of 3,2'-dinitrobiphenyl could not be determined because of its vague absorption spectrum. The introduction of the o-nitro group into 2-nitrobiphenyl and 4,4'-dinitrobiphenyl, to give 2,2'-dinitrobiphenyl and 2,4,2',4'-tetranitrobiphenyl, respectively, gives the similar ~kmax to nitrobenzene and m-dinitrobenzene respectively. Suzuki (1959) stated that a charge in spatial configuration about a single bond in any of the following 3 ways, depending on the degree of deviation from the coplanarity (Newmann, 1956) gave: (1) No change in the position of the absorption maximum but a decrease in the intensity. This effect is caused by relatively small twists. (2) A shift of the absorption maximum to shorter wavelength in addition to a decrease in the

105 TABLE 3 BLUE SHIFT OF X,..~ D U E TO THE NITRO SUBSTITUT I O N ON 2-POSITION OF BIPHENYLS Parent Compounds Biphenyl (BP) 2-NBP 3-NBP 4-NBP 4,2'-DNBP 4,3'-DNBP 4,4'-DNBP 4~4'-DN BP

2-Substituted Xm~ (rim)

Compounds

247 232 246 295 261 270 295 295

2-NBP 2,2'-DNBP 3,2'-DNBP 4,2'-DNBP 2,4,2'-TN BP 2,4,3'-TNBP 2,4,4'-TNBP 2,4,2',4'-TNBP

X,I~ (nm) 235 254 a 248 b 261 236 250 273 232 ~

/

1O

/

/

Xmax shift (nm) - 12 +22 a +2 b - 34 - 25 - 20 - 22 - 63 ~

a The Xmax of 2,2'-DNBP is almost similar to that of nitrobenzene (252 nm). b The exact Xmax of 3,2'-DNBP could not be determined because of its vague absorption spectrum. The Xmax of 2,4,2'A'-TNBP is almost similar to that of m-nitrobenzene (229 nm).

intensity. This effect is caused by larger twist than the first effect. (3) When the twist is large enough to eliminate almost completely the ~--~r interaction across the twisted bond, the spectrum is similar to the sum of the spectra of the component parts of the molecule on either side of the bond. In our experiments, the above effects were observed by the introduction of the o-nitro group into nitrobiphenyls. Fig. 1 shows the polarograms of 2-nitro-, 4nitro- and 4,4'-dinitrobiphenyl. Polarographic half-wave potentials of 2-nitro-, 4-nitro- and 4,4'dinitrobiphenyl were - 1 . 2 0 mV, - 1 . 0 8 mV and - 0 . 9 7 mV of first H 2 reduction potential, respectively. Apparently, 4-nitrobiphenyl was more reducible than the 2-isomer and further introduction of a nitro group at the 4'-position of 4-nitrobiphenyl (4,4'-dinitrobiphenyl) facilitates the initial reduction of the nitro group, but the reduction of the secondary nitro group was not observed by charging up to - 2.0 mV in the polarogram. The above results coincided with the results of Vance et al. (1985) and Sundvall et al. (1984). In the paper of Vance et al., 2,7-dinitrofluorene (2,7D N F ) was a strong mutagen in Salmonella typhimurium TA98 and TA98NR in the presence and absence of $9, and the potent mutagenic activity of 2,7-DNF arises from the metabolic

o

0,5

/

-i.0

!'/

-1.15 potet%tial

-210 ( mV vs Ag/AgCl )

Fig. i. Polarograms of 2-nitro-, 4-nhro- and 4,4'-dinitrobiphenyls. Polarograms were measured by P-1100-type polarograph (Yanagimoto Seisaku-sho, Kyoto, Japan). Tetraethylammonium perchlorate (0.1 M) in DMSO was used as blank solution. Conditions: mode: TAST; scan rate: 5 mV/sec: drop time: 1.5 sec; electrode: D.M.E. (h - 70 cm); reference electrode: Ag/AgCI; auxiliary electrode: Pt wire. Samples (ca. 1 mM) were dissolved in blank solution. Sample solution was degassed by the bubbling of N 2 gas before measurement of the polarogram. - - , 2-NBP; . . . . . . , 4-NBP; . . . . . , 4,4'DNBP.

activation of only one of the nitro groups, the unmetabolized nitro group serving to exert a strong electron-withdrawing effect on the aromatic structure. It is possible that the electron-withdrawing effect, due to a nitro group, may facilitate the initial reduction of a nitro group and increase the stability of the hydroxylamine, both of which would be expected to increase the potential for genetic damage. Sundvall et al. (1984) reported the mutagenicity on Salmonella typhimurium of nitrobenzoic acids and nitrotoluenes. In their paper, a different mutagenic effect on strain TA100 can be seen between isomers of the nitrobenzoic acids. The meta and para positions of the nitro group favor mutagenicity. An exception is 2,4-dinitrobenzoic acid, which has one para- and one ortho-oriented nitro group. The molecule is not planar because of steric repulsion between the nitro and carbonyl groups. A tentative explanation for its low mutagenicity would involve the assumption that only a fully planar nitroaromatic compound shows a high affinity to the nitro-reducing enzymes. Therefore, the presence of a nitro group at the 4-position in nitrobiphenyl maintained the planar-

106 ity of biphenyl owing to its electron-withdrawing effect, and the presence of a nitro group at the 2-position may cause the twisting form of the biphenyl at the C - C single bond. It is suggested that the p l a n a r form of n i t r o b i p h e n y l was advantageous not only for intercalation to D N A but also for high affinity to the nitro-reducing enzymes. Although, 2,4,2',4'-tetranitrobiphenyl has strong mutagenic potency in TA98, T A 9 8 N R and T A 9 8 / 1,8DNP 6, its molecule is not p l a n a r from its spectrum (Table 3). The above results were not convincingly explained as concerning only the position of the nitro group on the b i p h e n y l structure. King and King (1953) described the reaction of 2,2'-dinitrobiphenyl with sodium disulfide in ethanol, which gave benzo[c]cinnoline N-oxide together with 2 - a m i n o - 2 ' - n i t r o b i p h e n y l . Two benzo[c]cinnoline N-oxide derivatives were tested in TA98 and T A 9 8 N R (Table 1). 2-Nitro[c]cinnoline 6-oxide, which was synthesized from the n i t r a t i o n of benzo[c]cinnoline N-oxide as described by Barton a n d Cockett (1962), was mutagenic in both TA98 and T A 9 8 N R , whereas benzo[c]cinnoline N-oxide was inactive. The above results suggest that 2,4,2',4'- a n d 2,4,2',6'-tetranitrobiphenyl may reduce to b e n z o [ c ] c i n n o l i n e - t y p e c o m p o u n d s , whose molecules are planar, by a nitroreductase other than the classical nitroreductase a n d / o r other reducing enzyme system in T A 9 8 N R , a n d the p - n i t r o groups in 2,4,2',4'-tetranitrobiphenyl were necessary for its mutagenic potency.

Acknowledgments The authors are grateful to Prof. B.N. Ames, University of California, Berkeley, CA, who kindly supplied Salmonella typhimurium strain TA98, and to Prof. H.S. Rosenkranz, Case Western Reserve University, Cleveland, OH, who kindly supplied Salmonella tvphimurium strains T A 9 8 N R and T A 9 8 / 1 , 8 D N P o . We also wish to thank Ms. H. Takahashi, Ms. Y. Fujii and Ms. T. H o u d a for their excellent technical assistance in this investigation.

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