Mutation Research, 263 (1991) 13-19

13

© 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 016579929100025D MUTLET 0482

Mutagenicity of 4-nitrodiphenyl thioether-derived products and their potential metabolites T.R. Juneja, Baljeet Kaur and R.L. Gupta Department of Pharmaceutical Sciences, Panjab University, Chandigarh 160014 (India) (Received 26 September 1990) (Revision received 9 November 1990) (Accepted 14 December 1990)

Keywords: Nitrated diphenyl thioether; Salmonella typhimurium TA98; Potential metabolites; Mutagenicity

Summary 4-Nitrodiphenyl thioether (4-NO2TE) and its various potential metabolites 4-nitroso- (4-NOTE), 4-hydroxylamino- (4-NHOHTE), 4-hydroxyacetylamino- [4-N(OH)ACTE], 4-acetoxy-acetylamino- [4-N(OAc)ACTE], 4-amino- (4-ATE) and 4-acetamido- (4-AATE) were tested for their mutagenicity toward Salmonella typhimurium TA98 in the presence and absence of $9 activation system. 4-NO2TE, 4-NOTE, 4-NHOHTE and 4-N(OAc)ACTE were direct-acting mutagens with 4-NHOHTE having the highest activity. With $9 mix, the mutagenicity of 4NO2TE almost doubled and that of 4-NOTE and 4-N(OAc)ACTE decreased. In TA98NR, 4-NOTE induced higher mutations, while with 4-NHOHTE the number of revertants decreased. Both 4-NHOHTE and 4-N(OAc)ACTE showed decreased mutagenicity in TA98/1,8-DNP6. 4-ATE, 4-AATE and 4-N(OH)ACTE expressed mutagenicity only with $9 mix. 4-Nitrodiphenyl sulphoxide (4-NO2SO) and 4-nitrodiphenyl sulphone (4-NO2SO2), the sulphur-oxidised products of 4-NO2TE, were non-mutagenic as such and after addition of $9 activation system.

Nitroaromatics have widespread use as therapeutic agents and as intermediates in chemical industry. Their presence has also been detected in polluted urban air and diesel exhaust (Tokiwa and Ohnishi, 1986; Rosenkranz and Mermelstein, 1983). A number of these compounds have been found to be mutagenic/carcinogenic and this toxicity has long been recognised to be dependent Correspondence: Dr. T.R. Juneja, Department of Pharmaceutical Sciences, Panjab University, Chandigarh 160014 (India).

upon the metabolic conversion of the nitro function to its reduction products (nitroso, hydroxylamino) and various ester derivatives of hydroxylamine (Sternson, 1975; Tatsumi et al., 1986; Weisburger and Weisburger, 1973; Mangold and Hanna, 1982; Shinohara et al., 1986). Though nitrodiphenyl ether and its derivatives have been reported to be mutagenic (Miyauchi et al., 1984; Gupta et al., 1986), such studies on the corresponding nitrodiphenyl thioether seem to be lacking except a brief statement of the mutagenic activity of the antimalarial agent 4-aminoacyl-4'-nitrodi-

14

phenyl thioether (K611ing et al., 1986). In this paper, 4-nitrodiphenyl thioether and some of its likely metabolites such as nitroso, hydroxylamino, acetohydroxamic acid, O-acetyl ester of hydroxamic acid along with amino and acetamido have been tested for their mutagenicity in S. typhimurium TA98 and its derivatives. To examine the effect of sulphoxidation on mutagenicity, the sulphoxide and sulphone derivatives of 4-nitrophenyl thioethers were also tested. Materials and methods

Chemicals All compounds used in the present investigations were synthesised in the laboratory. The purity of the products was established by TLC and their identification was based on spectral analysis. The spectral data are compiled in Table 1. 4-NO2TE, 4-ATE, 4-AATE and 4-NO2SO2 were synthesised following the reported procedures (Kornblum, 1976; Raiziss et al., 1939; Auglage, 1944). 4-Hydroxylaminodiphenyl thioether (4-NHOHTE) was synthesised according to the method of Rising (1904). 4-Nitrosodiphenyl thioether (4-NOTE) was

obtained as a bright green crystalline product on oxidation of 4-NHOHTE with aqueous ferric chloride solution at -10°C, m.p. 38-40°C. 4-N-Acetoxy-N-acetylaminodiphenyl thioether [4-N(OAc)ACTE] was prepared by reacting 4-NHOHTE in CH2C12 with acetic anhydride. On removal of solvent under vacuum and purification on silica gel column a light brown oily mass was obtained. 4N-Hydroxy-N-acetylaminodiphenyl thioether [4-N(OH)ACTE] was obtained from its acetoxy derivative as mentioned above as a thick light brown viscous material on hydrolysis with methanolic ammonia and purification on silica gel column, and 4-NO2TE on treatment with H202 in glacial acetic acid gave 4-nitrodiphenyl sulphoxide (4-NO2SO) as white crystals, m.p. 98-100°C.

Mutagenicity assay procedure Bacterial strains used for the mutagenicity assay were S. typhimurium His - strains. TA98 was kindly provided by Prof. B.N. Ames, University of California (Berkeley, CA, U.S.A.) and TA98NR and TA98/1,8-DNP6 were furnished by Prof. H.S. Rosenkranz, Case Western Reserve University (Cleveland, OH, U.S.A.).

TABLE 1 SPECTRAL CHARACTERISTICS OF THE COMPOUNDS Compound

UV/vis (MeOH) (k . . . . nm)

4-NHOHTE

-

4-NOTE

345 (9772), 355 (7639)

4-N(OAc)ACTE

232 259 278 337 258 279

4-N(OH)ACTE

4-NO2SO

(10890), (13650), (12463), (3798) (18165), (18303)

262 (12165)

Vmax (cm - 1) 3312, 1580, 789 1578, 850, 686,

~H-NMR (CDCI3) (6)

3050, 1255, 1460, 740, 680

1797 (N-acetoxy), 1695 (amide), 1590, 1080, 745,687 3400-2800 (OH), 1680 (amide), 741,687 1520, 1342, 848, 686

MS (m/e) 217 (M +. ), 201 (M +. -O)

7.7 (d, 2H, J = 10 Hz), 7.45 (s, 5H), 7.2 (d, 2H, J = 10 Hz),

215 (M + ), 185 (M + -NO), 153 (185 -S)

7.4-7.9 (m, 9H), 2.4 (s, 3H), 2.2 (s, 3H)

301 (M +), 259 (M + -ketene)

7.4 (s, 9H), 5.4-5.7 (b, 2.1 (s, 3H) 8.2 (d, 2H, 7.7 (d, 4H, 7.2-7.6 (m,

IH),

259 (M +. ), 243 (M + -O), 217 ( M r -ketene)

J = 10 Hz), J = 10 Hz), 3H)

247 (M t )

15 The handling of the strains and experimental culture medium and other conditions of the assay have been described (Maron and Ames, 1983). For the actual assay, a 0.1-ml test solution in dimethyl sulphoxide (spectroscopic grade), 0.1 ml of a 16-h bacterial culture (equivalent to 2-4 × 10s cells) and 0.5 ml phosphate buffer, pH 7.4, or $9 mix was mixed with top agar containing 0.1 /~mole of histidine and 0.1 #mole of biotin and plated on minimal-glucose agar plates according to the procedure given in the test procedure. After incubating the plates for 2 days at 37°C, the revertant colonies were counted. For each dose the plates were set up in duplicate and the specific activities shown in Table 2 are the mean values of 6 independent experiments. The specific mutagenic activities were obtained from the slope of the linear section of the dose-response curves. The spontaneous revertant frequencies in the absence ( - ) and presence ( + ) of $9 per plates were: TA98 - 1 8 , +23; TA98NR - 31; and TA98/1,8-DNP6 - 14. Rat liver $9 fraction was prepared using male albino rats weighing 150-200 g, who received a single i.p. dose of Aroclor 1254 (500 mg/kg b.w.) diluted in corn oil (200 mg/ml) and who were killed on the 5th day after the injection. The $9 fraction was prepared from wet livers (3 ml of 0.15 M KC1/g wet liver) by centrifugation at 9000 × g for 10 min at 4°C. The post-mitochondrial supernatant ($9 fraction) of the liver was then stored in 2-ml quantities at -80°C until use. The $9 fraction was used to prepare S9 mix, which contained per ml: 0.1 ml of $9 fraction; MgC12'6H20 (8 #mole); KC1 (33 /~mole); sodium phosphate, pH 7.4 (100 /~mole); glucose-6-phosphate (5 /~mole) and NADP (4 /~mole).

-N(OAc)ACTE were tested in TA98/I,8-DNP6. 4-NO2TE, 4-NOTE, 4 - N H O H T E and 4-N(OAc)A C T E were found to be direct-acting mutagens with activity sequence 4 - N H O H T E > 4 - N ( O A c ) A C T E > 4 - N O T E > 4 - N O E T E having rev/nmole 76.95, 22.16, 14.60 and 1.60, respectively. In the presence of $9 mix, 4-NO2TE induced increased mutations and its activity almost doubled while a decrease in activity was found with 4-NOTE and 4-N(OAc)ACTE. 4 - N H O H T E and 4-N(OAc)A C T E were much less mutagenic in TA98/1,8DNP6 than in TA98:22.05 and 7.66 rev/nmole vs. 76.95 and 22.16. As reported for certain other nitrosoarenes, 4-NOTE also induced higher mutations (20.67 rev/nmole) in TA98NR than in TA98 in the presence or absence of activation system (14.60 and 11.1 rev/nmole respectively). In the absence of activation system the compound 4-NOTE showed a linear dose-response curve up to 80 nm/plate and thereafter it had antimicrobial activity while in the presence of activation system a linear relationship was noted up to 320 nm/plate and toxicity at doses higher than this. As expected 4-ATE, 4-AATE and 4-N(OH)ACTE were nonmutagenic as such and the activity was expressed only in the presence of activation system. 4-NO2SO2 and 4-NO2SO were non-mutagenic as such and in the presence of $9 mix. A likely secondary metabolite bis-(4,4'-thiophenoxy)-azoxybenzene expected to arise from 4 - N H O H T E was also found to be non-mutagenic in the presence or absence of activation system. 4-Amino-4'-nitrodiphenyl thioether (4-NO2ATE), a para-amino derivative of 4-NO2TE, was mutagenic but its activity was far less than that of 4-NOETE in the presence or absence of $9 mix (0.96 and 0.18 rev/nmole as against 3.31 and 1.60).

Results Discussion

The mutagenic activity of 4-NO2TE and its potential metabolites 4-NOTE, 4 - N H O H T E , 4-N(OH)ACTE, 4-N(OAc)ACTE, 4-ATE and 4-AATE was tested in strain TA98 in the presence and absence of the $9 metabolic activation system. Table 2 gives the dose-response results of the compounds tested. 4-NO2TE, 4 - N H O H T E and 4 -

The initial steps responsible for the carcinogenicity/mutagenicity of nitroaromatics have been considered through their relative ease of bioconversion to reductive products sequenced as: -NOE~NO~-NHOH~-NH2 (Andrews et al., 1983; Heflich et al., 1985). The hydroxylamine

16 TABLE 2 OBSERVED REVERTANT COUNT OF 4-NITRODIPHENYL S U L P H I D E AND ITS DERIVED PRODUCTS AND POTENTIAL METABOLITES IN Salmonella typhimurium TA98, TA98NR AND TA98/1,8-DNP6 Compound

Solvent control 4-NO2TE

4-NOTE

4-NHOHTE

4-N(OAc)ACTE

4-AATE

4-N(OH)ACTE

4-ATE

4-NOzATE

Amount

TA98

per plate (#M)

- $9

+ $9

TA98NR

0.0 0.04 0.16 0.32 0.64 rev/nM 0.02 0.08 0.32 rev/nM 0.01 0.02 0.04 rev/nM 0.04 0.08 0.16 0.32 rev/nM 0.08 0.32 0.64 rev/nM 0.04 0.08 0.16 rev/nM 0.08 0.32 1.28 rev/nM 0.02 0.08 0.32 rev/nM

18 356 +_ 25 513 _+ 37 560 +_ 42 1210 + 59 1.60 _+ 0.15 1140 +_ 90 1425 _+ 120 404 _+ 39 14.60 _+ 1.1 548 _+ 44 1704 _+ 133 3000 _+ 259 76.95 _+ 4.0 938 _+ 80 2012 + 180 4600 _+ 421 6960 +_ 551 22.16 _+ 2.1 40 _+ 2.1 18 +_ 2.0 18 +_ 4.0 0.0 25 _+ 2.0 30 _+ 3.0 49 +_ 3.0 0.19 _+ 0.12 7 _+ 2 37 _+ 3.0 NT 0.0 46 _+ 4.0 64 _+ 5.0 90 _+ 11 0.18 _+ 0.03

23 NT 343 +_ 29 943 _+ 79 2087 +_ 110 3.31 + 0.30 61 _+ 5 869 + 70 3511 _+ 266 11.1 + 1.0 NT NT NT 361 1410 2630 5224 16.59 400 661 1355 1.92 182 2007 3280 22.01 556 1558 3128 2.24 42 265 349 0.96

31 38 _+ 35 _+ NT 48 +

TA98/1,8-DNP6

4 5 6

1056 + 101 1859 +_ 155 317 _+ 24 20.67 _+ 2.0 425 +_ 38 1638 _+ 135 2248 _+ 230 58.23 +_ 4.1 NT NT NT NT

_+ 35 _+ 130 _+ 144 _+ 499 +_ 0.9 _+ 30 _+ 57 _+ 130 + 0.12 + 17 _+ 190 +_ 199 _+ 2.1 +_ 20 + 143 _+ 340 +_ 0.23 _+ 4.0 _+ 21 +_ 30 _+ 0.10

NT NT NT

14 122 _+ 9 175 + 16 NT 345 _+ 27 0.44 _+ 0.04 NT NT NT 112 _+ 10 340 _+ 25 868 + 70 22.05 _+ 2.0 151 + 10 576 _+ 46 1170 _+ 97 2400 _+ 210 7.66 _+ 0.7 NT NT NT

NT NT NT

NT NT NT

NT NT NT

NT NT NT

NT NT NT

NT NT NT

The expressed results are the mean _+ SD of data (n = 6). Significant differences between - $9 and + $9 were obtained as demonstrated by Student's t-test ( P < 0.05). Positive control: 4-nitroquinoline N-oxide (TA98) gave 160 revertants at dose 0.5 ~g. 2-Aminoanthracene (TA98) gave 1330 revertants at dose 5 ~g, in the presence of $9 mix.

considered cinogenic

to

be

the

metabolite

proximate may

further

fication reactions and in particular acetyl-CoA-dependent 4-N(OH)ACTE, toxyaminodiphenyl

enzymes

4-N(OAc)ACTE

mutagenic/car-

a t i v e s ( W i r t h et a l . , 1980; S a i t o et a l . , 1985). T h e

undergo

N-acetoxy

and

considered

to be the ultimate carcinogenic/muta-

esteri-

acetylation

by

N,O-diacetyl

to

produce

genic metabolites,

and

4-N-ace-

or N-acetylnitrenium

thioether 4-NH(OAc)TE

deriv-

derivatives

which may generate

may

be

nitrenium

ion respectively by the facile

loss of an acetate ion. Of the 2 competing

reac-

17 tions, in vivo conversion of the hydroxylamine derivative to N-acetoxy-N-acetylamino or N-acetoxyamino, the latter path could be considered to be more toxic due to the highly reactive nature of N-acetoxyarylamines as these are only transitory intermediates which decompose further during the course of reaction. In comparison, N-acetoxyacetylarylamines can be synthesised in the laboratory. N-Acetoxyarylamine in the cell could also probably arise directly from nitrosoarenes even non-enzymatically in the presence of the N A D P H / NADH reducing system and acetyl CoA. In synthetic experiments, we have observed the intermediate formation of N-acetoxyarylamine from nitrosoarenes by treatment with a synthetic model of N A D P H / N A D H and acetyl-CoA (Juneja et al., 1984). It has been further observed by us that Nacetoxyarylamine may undergo cleavages (i) heterolytic to generate nitrenium ion, and (ii) homolytic to generate amino radical, and (iii) o~elimination reaction to generate nitrene (Juneja and Dannenberg, 1975). The nature of the aromatic ring has been found to have a profound influence on the formation of these 3 electrophilic species (Pathak, 1987). The generation of Nacetoxyarylamine and its subsequent fate(s) in the cell may also be implicated in explaining the differences in biological activity of various nitroaromatics. 4-N(OAc)ACTE, in contrast to 4-Nacetoxy-N-acetylaminodiphenyl ether 4-N(OAc)ACE, was found to be a direct-acting mutagen with slightly decreased activity with $9 mix. 4-N(OAc)ACE has been reported to be mutagenic only in the presence of $9 activation system (Miyauchiet al., 1984). Thus the direct-acting mutagenicity of 4N(OAc)ACTE in comparison to 4-N(OAc)ACE could be due to the facile loss of an acetate ion from the former to generate an N-acylnitrenium ion. The presence of a sulphur bridge between the 2 rings in thioether vis-a-vis oxygen in ether seems to destabilise the N-acetoxy bond by increasing electron density through earlier delocalisation of the electron cloud from sulphur to the phenyl ring having an N-acetoxy bond. The decrease in mutagenic activity of 4-N(OAc)ACTE in the presence of $9 mix may be due to possible non-specific binding

of this reactive ester with nucleophilic components in $9. It is, however, puzzling to observe a great decrease in the mutagenic activity of this compound in TA98/1,8- DNP6 (7.66 rev/nmole vs. 22.16 in TA98). The N(OAc)AC derivative of fluorene has been reported to have almost the same mutagenicity in TA98 and its derivatives (McCoy et al., 1982). As with 2-hydroxylaminofluorene, 4N H O H T E was also found to have higher mutagenicity than the corresponding 4-N(OAc)ACTE (thought to be an ultimate reactive metabolite) and this large decrease in activity (76.95 to 22.16 rev/nmole) could also be explained by the possible syn-configuration (Z-conformation) of the N-acetylamino DNA adduct (McCoy et al., 1982). The S9-dependent increase in mutagenicity of 4-NOETE, as also found in a number of other nitroarenes such as 4-nitrobiphenyl ether (Miyauchi et al., 1984) and substituted 4-nitrobiphenyl ethers (Hirayama et al., 1990), could be accounted for by the existence of partial anaerobiasis in the plate containing active $9 nitroreductases. The slight decrease in the mutagenicity of 4-NOTE in the presence of $9 mix appears to be due to non-specific binding of the reactive nitroso group with nucleophilic residues of $9.4-ATE and 4-AATE as in 4-amino- and 4-acetylamino-biphenyl ether were mutagenic only after $9 activation and this seems to be due to their metabolic conversion to corresponding Noxidation products (-NNOH, N(OH)AC). The amide derivative (4-AATE) was less mutagenic than the corresponding amino 4-ATE and this behaviour has been observed in some aromatic amines and amides. It has been explained that the N-oxidised metabolite of amide, hydroxamic acid, needs an additional step for its conversion to the more active hydroxylamine by the deacylase enzyme system (Wirth et al., 1980). The sulphoxide 4-NO2SO and sulphone 4-NO2SO2 derivatives did not show mutagenic activity in the absence and presence of $9 mix. This absence of specific activity seems to be due to the paucity of electronic transmission between the 2 phenyl rings which is otherwise possible when they are connected through the sulphur bridge and not the oxidised sulphur present in sulphoxide and

18

sulphone derivatives as visualised by NMR (Hyne and Greidanus, 1969). The structures may be represented as: ®

(~)__

/7---~ . ~ ° ~_~

The expression of biological activity in this series of compounds appears to be linked with the planar structure of the molecule and the conjugation of the 2 phenyl rings through the sulphur bridge, thus facilitating the possible increase of electron density around the nitro function and, in turn, its reduced product hydroxylamine and its esters. Heterolytic N-O cleavage in such derivatives is expected to be facile to generate electrophilic nitrenium ion. This argument is further strengthened as we found 4 - N H O H T E to be highly unstable, decomposing even by storing overnight at - 80°C. Further, a different product was always obtained in our crystallisation attempts. The direct mutagenicity of 4-N(OAc)ACTE in contrast to 4-N(OAc)ACE could also be explained to the easy cleavage of the N-OAc bond in the former. It is interesting to observe the sequence of mutagenic activity among the 3 compounds having 2 phenyl rings with a hetero atom (S or O or N) bridge in between and a nitro function attached at the p a r a position in one phenyl ring, in TA98 as 4-NOzTE > 4-NO2E > 4-NO2A (4-nitrodiphenylamine) with 1.6, 0.18 and 0.00 rev/nmole respectively. 4-NO2A was inactive as such and after metabolic activation. Going across the periodic table one finds that the sequence of the hardness of the bases is N > O > S as opposed to softness S > O > N. Thus the delocalisation of the electron from the -S- bridge into the ring is expected to be more than -O- which in turn is more than the -Nbridge and this sequence of electron transfer of the bridged atom is in exactly the same order as their mutagenicity sequence. This indicates that generation of electron density around the nitro function through cross-conjugation between the 2 phenyl rings and an atom between may play an important role in determining the mutagenicity of such compounds. However, we are collecting more details in this direction.

To have useful drugs or chemicals likely to be consumed by human beings among the series of compounds with a nucleus of diphenyl thioether and various nitrogen functions, it is suggested that safer drugs/chemicals devoid of possible mutagenic/carcinogenic activity could be achieved if the conjugation between the 2 phenyl rings with a sulphur bridge is hindered. References Andrews, P.A., D. Bryant, S. Vitakunas, M. Gouin, G. Anderson, B.E. McCarry, M.A. Quilliam and D.R. McCalla (1983) Metabolism of nitrated polycyclic aromatic hydrocarbons and formation of DNA adducts in Salmonella typhimurium, in: M. Cook and A.J. Dennis (Eds.), Polynuclear Aromatic Hydrocarbons - Formation, Metabolism and Measurement, Battelle Press, Columbus, OH, pp. 89-98. Auglage, V. (1944), Beilstein Handbuch der organischer Chemie, Springer-Verlag, Berlin, ed. EII 6, p. 311. Gupta, R.L., D.K. Dey and T.R. Juneja (1986) Mutagenicity of 4-nitrodiphenyl ether and its metabolites, Toxicol. Lett., 34, 13-21. Heflich, R.H., P.C. Howard and F.A. Beland (1985) l-Nitrosopyrene - an intermediate in the metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538, Mutation Res., 149, 25-32. Hirayama, T., K. Iguchi and T. Watanabe (1990) Metabolic activation of 2,4-dinitrobiphenyl derivatives for their mutagenicity in Salmonella typhimurium TA98, Mutation Res., 243,201-206. Hyne, J.B., and J.W. Greidanus (1969) Nuclear magnetic resonance study of intramolecular electronic effects in diphenyl sulfides, sulfoxides, and sulfones, Can. J. Chem., 47, 803-812. Juneja, T.R., and H. Dannenberg (1975) Attempts to prepare aromatic O-acylhydroxylamines. II. Dehydrohalogenation of 1- bromo-4- acetoxy-imino- 1,2,3,4-tetrahydrophenanthrene by 1,8-diazabicyclo[5,4,0]-undec-7-ene (DBU), Tetrahedron, 31,701-708. Juneja, T.R., A. Ojha and R.L. Gupta (1984) NAD(P)H and acetyl-CoA models. Part 1: reaction of nitrosobenzene with 1,1 '-diacetyl- 1,1' ,4,4' -tetrahydro-4,4' -bipyridine, Indian J. Chem., 23B, 60-66. K611ing, V.H., A. Haberkorn and B. Herbold (1986) Untersuchungen an malariawirksamen ~-Aminoacyl-Verbindungen, Drug Res., 36, 230-233. Kornblum, N., L. Cheng, R.C. Kerber M.M. Kestner, B.N. Newton, H.W. Pinnick, R.G. Smith and P.A. Wade (1976) Displacement of the nitro group of substituted nitrobenzenes a synthetically useful process, J. Org. Chem., 41, 1560-1568. Mangold, B.L.K., and P.E. Hanna (1982) Arylhydroxamic acid

19 N,O-acyltransferase substrates. Acetyl transfer and electrophilic generating activity of N-hydroxy-N-(4-alkyl-, 4-alkenyl-, and 4-cyclohexylphenyl)acetamides, J. Med. Chem., 25,630-638. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 177-215. 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, 1362-1367. Miyauchi, M., Y. Takou, M. Watanabe and T. Uematsu (1984) Mutagenic activity of possible metabolites of 4-nitrobiphenyl ether, Chem.-Biol. Interact., 51, 49-62. Pathak, D.P. (1987) Reaction of Arylnitroso and N-Nitrosamines with 1,1'-Diacetyl-l,1 ',4,4'-tetrahydro-4,4'-bipyridine (DTB) - An in vitro NAD(P)H and Acetyl-CoA Model, Ph.D. Thesis, Panjab University, Chandigarh. Raiziss, G.W., L.W. Clemence, M. Severac and J.C. Moetsch (1939) Chemistry and chemotherapy of 4,4'-diaminodiphenylsulfone, 4-amino-4'-hydroxydiphenylsulfone and related compounds, J. Am. Chem. Soc., 61, 2763-2765. Rising, A. (1904) Uber die Methyl und Athyl~ither des Oxyphenylhydroxylamines und die daraus dargestellten Azoxyverbindungen, Ber. Dt. Chem. Ges., 37, 43-47. Rosenkranz, H.S., and R. Mermelstein (1983) Mutagenicity and genotoxicity of nitroarenes - all nitro-containing chemicals were not created equal, Mutation Res., 114, 217-267 (and references therein). Saito, K., A. Shinohara, T. Kamataki and R. Kato (1985) Metabolic activation of mutagenic N-hydroxylamines by O-

acetyltransferase in Salmonella typhimurium TA98, Arch. Biochem. Biophys., 239, 286-295. Shinohara, A., K. Saito, Y. Yamazoe, T. Kamataki and R. Kato (1986) Acetyl coenzyme A dependent activation of N-hydroxy derivatives of carcinogenic arylamines - mechanism of activation, species difference, tissue distribution and acetyl donor specificity, Cancer Res., 46, 4362-4367. Sternson, L.A. (1975) Detection of arylhydroxylamines as intermediate in the metabolic reduction of nitro compounds, Experientia, 31,268-269. Tatsumi, K., S. Kitamura and N. Narai (1986) Reductive metabolism of aromatic nitro compounds including carcinogens by rabbit liver preparations, Cancer Res., 46, 1089-1093. Tokiwa, H., and Y. Ohnishi (1986) Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol., 17, 23-60 (and references therein). Weisburger, J.H., and E.K. Weisburger (1973) Biochemical formation and pharmacological, toxicological and pathological properties of hydroxylamine and hydroxamic acids, Pharmacol. Rev., 25, 1-26. Wirth, P.J., E. Dybing, C. von Bahr and S.S. Thorgeirsson (1980) Mechanism of N-hydroxyacetylarylamine mutagenicity in the Salmonella test system - metabolic activation of Nhydroxyphenacetin by liver and kidney fractions from rat, mouse, hamster and man, Mol. Pharmacol., 18, 117-127. Communicated by J.M. Gentile