Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds

Article abstract of DOI:10.1016/S1383-5718(01)00140-1

Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100 compared with that of the corresponding hydroxylamines and the previously studied nitroarenes. A remarkable correspondence of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds. This effect could be observed in TA98 and TA100. It was only marginally dependent on the metabolical activation by rat liver S9-mix. Even the presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine species. The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine species under the test conditions. It seems that enzymes are not responsible for that reduction step, because sterical crowding near the functional group does not influence that behaviour. The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence the mutagenicity of an aromatic nitro compound by such a group. A substituent near the functional group (ortho-position) disturbs the enzymatic reduction of the nitro group, because 3-tert-butyl-4-hydroxylaminobiphenyl and its corresponding nitroso compound are highly mutagenic, whereas 3-tert-butyl-4-nitrobiphenyl was previously shown to be inactive even after addition of S9-mix. In contrast, 4′-tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group "far away" from the hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in that position. In addition, the substance shows only little cell toxicity even at higher concentrations. Both effects could be due to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster degradation of the hydroxylamine or a hindered interaction between that substance and the cells.

Full text of DOI:10.1016/S1383-5718(01)00140-1

Mutation Research 491 (2001) 183–193
Mutagenicity in Salmonella typhimurium TA98 and TA100 of
nitroso and respective hydroxylamine compounds
Torsten Haacka, Lothar Erdingerb, Gernot Bochea,∗
a
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein Straße, 35032 Marburg, Germany
b
Hygieneinstitut Heidelberg, Im Neuenheimer Feld, 69120 Heidelberg, Germany
Received 24 October 2000; received in revised form 16 January 2001; accepted 17 January 2001
Abstract
Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100
compared with that of the corresponding hydroxylamines and the previously studied nitroarenes. A remarkable correspondence
of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds. This effect could
be observed in TA98 and TA100. It was only marginally dependent on the metabolical activation by rat liver S9-mix. Even the
presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably
influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine
species. The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine
species under the test conditions. It seems that enzymes are not responsible for that reduction step, because sterical crowding
near the functional group does not influence that behaviour.
The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence
the mutagenicity of an aromatic nitro compound by such a group. A substituent near the functional group (ortho-position)
disturbs the enzymatic reduction of the nitro group, because 3-tert-butyl-4-hydroxylaminobiphenyl and its corresponding
nitroso compound are highly mutagenic, whereas 3-tert-butyl-4-nitrobiphenyl was previously shown to be inactive even
after addition of S9-mix. In contrast, 40-tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group “far away” from the
hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in
that position. In addition, the substance shows only little cell toxicity even at higher concentrations. Both effects could be due
to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster
degradation of the hydroxylamine or a hindered interaction between that substance and the cells. © 2001 Elsevier Science
B.V. All rights reserved.
Keywords: Salmonella; Nitroaromatics; Alkyl substituent; Metabolites; Hydroxylamines; Nitroso compounds
1. Introduction
dye and plastic production, although some of these
chemicals are known to be mutagenic and carcino-
genic [1]. One possibility to decrease the mutagenicity
of a substance is a purposeful structural modification
which should be as insignificant as possible in order
to keep the desired properties of the compound. We
recently reported that the mutagenicity of certain aro-
matic nitro compounds in Salmonella typhimurium
A large number of aromatic amines and nitro com-
pounds are still used in industrial processes, such as
∗ Corresponding author. Tel.: +49-6421-282-2030;
fax: +49-6421-282-8917.
E-mail address: boche@chemie.uni-marburg.de (G. Boche).
1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(01)00140-1

184
T. Haack et al. / Mutation Research 491 (2001) 183–193
TA98 and TA100 can be significantly reduced by
the introduction of bulky alkyl groups either near the
functional group, or far away from it [2,3]. In order
to use that kind of structural modification as a general
strategy, some further information should be obtained
about the effect that is carried out by bulky alkyl
groups, which is the purpose of this work.
The mutagenicity of aromatic nitro compounds is
thought to be induced by a chemical modification
of the deoxyribonucleic acid (DNA), resulting from
an electrophilic attack of an activated nitrogen inter-
mediate, as, e.g. N-acetoxy-4-aminobiphenyl, at the
purine bases of DNA, above all at the C8-position
of deoxyguanosine [4,5]. This and other reactive
electrophiles are supposed to be generated by two
reduction steps from a nitroarene affording first the
corresponding nitroso compound, and subsequently
the hydroxylamine, which is further activated by
O-acetylation, O-sulfatation or O-protonation [6].
Replication failures occurring with the modified DNA
are thought to be finally responsible for the tumour
induction.
2. Materials and methods
2.1. Instrumentation
NMR spectra were recorded on a Bruker spec-
trometer ARX200 and are referenced to tetramethyl-
silane as an internal standard. Mass spectra were
obtained on a Varian MAT CH-7-A (EI, 70 eV).
Elementary analyses were recorded by a Heraeus
rapid Elementaranalysator. The melting points are
uncorrected.
2.2. Chemicals
The aromatic nitro compounds 4 and 7 and the
nitroso compounds 2, 5, 8 were synthesised according
to Klein et al. [2,3], and 2-nitrosofluorene (11) was
prepared in analogy to that procedure. 4-Nitrobiphenyl
(1), 2-nitrofluorene (10) and 2-nitronaphthalene were
purchased from Aldrich chemicals. The hydroxy-
lamines 3, 6, 9 and 12 were prepared by reduction of
1, 4, 7 and 10, respectively, with ammonium chloride
and zinc dust. The hydroxylamine 14 and the nitroso
compound 13 were prepared according to literature
procedures [9,10]. All samples that were tested in the
Ames assay were repeatedly purified by recrystallisa-
tion until they contained no more impurities as judged
by TLC and NMR.
We have already shown that 40-tert-butyl-4-nitro-
biphenyl (7) (40-tBu-4-NBp) and the corresponding
40-tert-butyl-4-nitrosobiphenyl (8) (40-tBu-4-NOBp)
are almost non-mutagenic even after activation with
S9-mix [3]. In contrast, 3-tert-butyl-4-nitrosobiphenyl
(5) (3-tBu-4-NOBp), which is the corresponding nit-
roso compound of the non-mutagenic 3-tert-butyl-4-
nitrobiphenyl (4) (3-tBu-4-NBp), is highly mutagenic
[2]. Hence, different reasons seem to be responsible
for mutagenicity, or its suppression, in the two cases.
Therefore, we prepared the corresponding hydroxyl-
amines, 40-tert-butyl-4-hydroxylaminobiphenyl (9)
(40-tBu-4-NOHBp) and 3-tert-butyl-4-hydroxylami-
nobiphenyl (6) (3-tBu-4-NOHBp), as further exam-
ples for modification of the directly acting mutagens
with a sterical disturbance near the functional group,
and far away from it, respectively. We compared their
mutagenicity with the activity of their nitroso and
their nitro analogues in order to get further insight
into the role of the reduction processes. For compar-
ison, we also tested the corresponding non-alkylated
hydroxylamines, nitroso and nitro compounds of
4-nitrobiphenyl (1–3) and some other important mu-
tagenic nitroarenes, i.e. of fluorene (10–12) and naph-
thalene (13 and 14), which have already been shown
to be mutagenic by McCann et al. [7] (Fig. 1).
2.2.1. 4-Hydroxylaminobiphenyl (4-NHOHBp) (3)
A suspension of 4-nitrobiphenyl (1) (10 g, 50.2
mmol) and ammonium chloride (4.6 g, 86.0 mmol) in
200 ml methanol and 20 ml water was preheated to
50◦C. Zinc dust (15 g, 229 mmol) was then added in
small portions over a period of 2 min. The mixture
was vigorously stirred. After 1 min the suspension
started boiling, and after further 5 min of stirring the
mixture was quickly filtered and poured on ice. The
precipitate was extracted with ether, washed with
water and brine, dried over magnesium sulphate and
evaporated to dryness. The crude product, which
contained some amine, was then recrystallised from
trichloromethane to yield pure 3 (4.61 g, 48%). Pale
yellow plates, 1H-NMR (CDCl3, 200 MHz): δ = 8.40
(s, 2H, NHOH), 7.60–7.28 (m, 7H, Harom), 6.92 (d,
3
2H, J = 8.3 Hz, H3). 13C-NMR (CDCl3, 50 MHz):
δ = 151.8, 140.6, 131.2, 129.0, 127.0, 126.4, 126.0,
113.5. MS (70 eV): m/z (%): 185 (M+, 67), 168 (100),

T. Haack et al. / Mutation Research 491 (2001) 183–193
185
Fig. 1. Compounds studied in this work (2, 3, 5, 6, 8, 9, 11, 12, 13, 14) together with others used for comparison (1, 4, 7, 10). Letter ‘a’
in superscript indicates mutagenicity data taken from [2] and [3].
152 (49), 141 (53), 115 (36). Calculated: C 77.81, H
5.99, N 7.56; found: C 77.74, H 5.95, N 7.37.
133.1, 126.8, 125.3, 125.0, 120.2, 118.8, 112.1, 109.8,
36.6. Calculated: C 79.16, H 5.62, N 7.10; found: C
78.98, H 5.62, N 7.11.
2.2.2. 2-Hydroxylaminofluorene (2-NHOHF) (12)
This compound was prepared from 10 in analogy
to the procedure described for 3.
2.2.3. 3-tert-Butyl-4-hydroxylaminobiphenyl
(3-tBu-4-NOHBp) (6)
Yellow crystals, 1H-NMR (CDCl3, 200 MHz):
δ = 8.43 (s, br, 2H, NHOH), 7.75–7.67 (m, 2H, H5,
To a solution of 3-tert-butyl-4-nitrobiphenyl (4)
(0.3 g, 1.17 mmol) in 10 ml diethyl ether was added
ammonium chloride (0.15 g, 2.8 mmol) and then
water until all solid had dissolved. The two-phase
system was vigorously stirred and charged with
zinc dust (0.8 g, 12.2 mmol). The reaction was mon-
itored by TLC (diethyl ether/hexanes 1:2). When
3
H8), 7.51 (d, 1H, J = 7.4 Hz, H4), 7.33 (dd, 1H,
3
3
3J = 7.5 Hz, J = 7.5 Hz, H6), 7.24 (dd, 1H, J =
3
7.5 Hz, J = 7.5 Hz, H7), 7.10 (s, 1H, H1), 6.89 (d,
1H, J = 7.5 Hz, H3), 3.85 (s, 2H, CH2). 13C-NMR
3
(DMSO, 50 MHz): δ = 151.9, 144.1, 142.3, 141.9,

186
T. Haack et al. / Mutation Research 491 (2001) 183–193
the reaction had finished the reaction mixture was
partitioned between 20 ml diethyl ether and 20 ml
water. The aqueous phase was repeatedly extracted
with ether and the combined organic layer was dried
over magnesium sulphate and carefully evaporated to
dryness. The crude product, which contained some
amine, was then recrystallised from a mixture of
ether and hexanes at −50◦C to yield 6 (112 mg,
40%).
m/z (%): 183 (M+, 85), 169 (19), 153 (100), 128 (18),
102 (11), 77 (15), 28 (68).
2.2.7. 2-Nitrosofluorene (2-NOF) (11)
This compound was prepared in analogy to
the oxidation procedure described in [2] using
Mo[O2]2O·HMPT·H2O.
Green crystals, mp 77◦C. 1H-NMR (CDCl3,
3
200 MHz). δ = 8.21 (d, 1H, J = 8.0 Hz, H3), 7.97
1
3
Colourless crystals, mp 75–77◦C (dec.). H-NMR
(d, 1H, J = 8.0 Hz, H4), 7.96–7.87 (m, 1H), 7.79
(CDCl3, 200 MHz): δ = 7.45–7.15 (m, 7H, Harom),
6.60 (d, 1H, 3J = 8.0 Hz, H3), 3.61 (s, br, 2H,
NHOH), 1.36 (s, 9H, –C(CH3)3). 13C-NMR (CDCl3,
50 MHz): δ = 144.0, 141.8, 133.8, 131.5, 128.6,
126.5, 126.1, 125.6, 125.6, 118.1, 34.4, 29.6. MS
(70 eV): m/z (%): 225(M+ –OH, 86), 210 (100), 195
(14), 182 (28), 178 (18), 169 (21), 126 (25). Calcu-
lated: C 79.63, H 7.94, N 5.80; found: C 79.44, H
8.00, N 6.16.
(s, 1H, H1), 7.64–7.54 (m, 1H, Harom), 7.48–7.41 (m,
2H, Harom). 4.00 (s, 2H, CH2). 13C-NMR (CDCl3,
50 MHz): δ = 166.0, 148.5, 145.8, 143.7, 139.6,
128.9, 127.4, 125.4, 124.4, 121.7, 120.1, 115.4, 37.0.
Calculated: C 79.98, H 4.65, N 7.17; found: C 79.94,
H 4.79, N 7.00.
2.2.8. 40-tert-Butyl-4-nitrosobiphenyl
(40-tBu-4-NOBp) (8)
This compound was prepared according to [3].
1
2.2.4. 40-tert-Butyl-4-hydroxylaminobiphenyl
(40-tBu-4-NOHBp) (9)
Dark green crystals, mp 109◦C. H-NMR (CDCl3,
3
200 MHz): δ = 7.95 (d, 2H, J = 8.6 Hz, H2), 7.81
3
3
This compound was prepared from 7 in analogy to
the procedure described for 6.
(d, 2H, J = 8.6 Hz, H3), 7.62 (d, 2H, J = 8.6 Hz,
H20), 7.51 (d, 2H, J = 8.7 Hz, H30), 1.37 (s, 9H,
3
1
Colourless crystals, H-NMR (CDCl3, 200 MHz):
tBu). 13C-NMR (CDCl3, 50 MHz): δ = 165.0, 152.3,
147.9, 136.1, 127.5, 127.1, 126.1, 121.7, 34.7, 31.2.
Calculated: C 80.30, H 7.16, N 5.85; found: C 79.95,
H 7.13, N 5.73.
3
δ = 7.60–7.42 (m, 6H, Harom), 7.05 (d, 2H, J =
8.6 Hz, H2), 6.01 (s, br, 2H, NHOH), 1.35 (s, 9H,
–C(CH3)3). 13C-NMR (CDCl3, 50 MHz): δ = 147.7,
146.7, 135.9, 133.2, 125.5, 124.3, 123.7, 113.0, 32.5,
29.4. MS (70 eV): m/z (%): 225 (M+ –OH, 80),
210 (100), 194 (14), 182 (13), 69 (11). Calculated:
C 79.63, H 7.94, N 5.80; found: C 79.44, H 8.04,
N 5.55.
2.2.9. 3-tert-Butyl-4-nitrosobiphenyl (3-tBu-4-NOBp)
(5)
This compound was prepared according to [2].
Green crystals, mp 87◦C. 1H-NMR (CDCl3,
200 MHz): δ = 7.88 (s, 1H, H5), 7.60–7.31 (m, 6H,
3
2.2.5. 2-Hydroxylaminonaphthalene (2-NHOHNp)
(14)
This compound was prepared according to [9].
Colourless crystals, mp 135–137◦C (dec.). Calcu-
lated: C 75.45, H 5.70, N 8.80; found: C 75.12, H
5.54, N 9.12.
Harom), 6.08 (d, 1H, J = 8.4 Hz, H2), 1.82 (s, 9H,
tBu). 13C-NMR (CDCl3, 50 MHz): δ = 164.6, 152.9,
147.7, 139.9, 129.0, 128.6, 127.4, 126.5, 124.5, 107.4,
37.1, 33.4. Calculated: C 80.30, H 7.16, N 5.85;
found: C 80.01, H 7.32, N 5.65.
2.2.10. 2-Nitrosonaphthalene (2-NONp) (13)
2.2.6. 4-Nitrosobiphenyl (4-NOBp) (2)
This compound was prepared according to [10].
Grey crystals (green melt), mp 62◦C. 1H-NMR
(CDCl3, 200 MHz): δ = 9.49 (s, 1H, H1), 8.27 (d, 1H,
3J = 8.8 Hz, H3), 7.85–7.64 (m, 4H, Harom), 6.98 (d,
This compound was prepared according to [2].
Green crystals, mp 74◦C. 1H-NMR (CDCl3,
3
200 MHz): δ = 7.97 (d, 2H, J = 8.6 Hz, H2), 7.81
(d, 2H, 3J = 8.6 Hz, H3), 7.64−7.46 (m, 5H, Harom).
13C-NMR (CDCl3, 50 MHz): δ = 165.0, 148.1,
139.1, 129.1, 128.9, 127.8, 127.5, 121.6. MS (70 eV):
1H, J = 8.8 Hz, H4). 13C-NMR (CDCl3, 50 MHz):
3
δ = 163.7, 137.3, 136.4, 132.9, 131.0, 130.5, 129.0,
128.1, 127.7, 108.2.

T. Haack et al. / Mutation Research 491 (2001) 183–193
187
2.3. Mutagenicity tests
decreasing effect on the cell toxicity at higher con-
centrations of the compound, especially in the case
of the hydroxylamines 3 (see Fig. 2), 12 (see Fig. 3)
and 14 (see Table 1).
Although the same results could be qualitatively de-
termined in TA100 for the fluorene compounds 10–12
and the naphthalene derivatives 13 and 14, the strong
influence of cell toxicity does not allow any reliable
quantitative statement in that tester strain.
The compounds were tested as previously des-
cribed [2] at the Hygiene-Institut of the University
of Heidelberg, Germany, following the standard pro-
cedure described by Maron and Ames [8]. The tests
were carried out three times per sample and the re-
sulting revertant numbers were averaged. The relation
between the revertant number of a substrate and the
negative control (pure DMSO or THF, respectively)
was declared as the induction factor. The decrease of
mutagenicity after the peak of induction that can be
observed in most cases is due to a growing influence
of cell toxicity at higher doses.
3.2. Alkylated compounds
Regarding the mutagenic activity of 40-tBu-4-
NOHBp (9), 3-tBu-4-NOHBp (6), and their nitroso
analogues 5 and 8, we found considerable differences
with regard to the position of the tert-butyl groups,
see Table 2.
3. Results
Although 3-tBu-4-NBp (4) is not mutagenic at all, in
the presence of S9-mix or without, the corresponding
hydroxylamine 6 and nitroso compound 5 are both
heavily mutagenic. The curves in TA98 are shown in
Fig. 4. TA100 leads to comparable results.
It should be noted that the curves with and without
S9-mix are almost the same. In both cases, cell toxicity
is more important for the hydroxylamine 6. In contrast
to these results, the 40-alkylated biphenyls 7, 8 and 9
show a different behaviour, see Fig. 5.
3.1. Non-alkylated compounds
4NOBp (2) and 4NHOHBp (3), which have already
been found to be directly mutagenic in Salmonella
typhimurium TA8 [7], are directly acting mutagens in
TA98 and TA100, see Table 1.
4-NBp (1) was earlier shown to be a weak direct
mutagen in TA98 and TA100 [2,3]. Comparing the
dose-response curves of 1, 2 and 3, we found the
curves of 4-NOBp (2) to be very similar to those of
4-NHOHBp (3), but not to the results of 4-NBp (1).
Especially in TA100, with and without S9-mix, there is
a remarkable equality between the curves of 4-NOBp
(2) and 4-NHOHBp (3), see Fig. 2. The tests in TA98
show comparable results, but cell toxicity is not as
critical as in TA100.
In the case of the fluorene compounds 10–12 we
also found the same behaviour in TA98 and TA100.
Again, the dose-response curves of the hydroxylamine
12 and the nitroso compound 11 are very similar, while
the nitro compound 10 behaves differently. High cell
toxicity is found at higher doses, especially in the ab-
sence of S9-mix, see Fig. 3. A comparable similar-
ity was furthermore found between 2-NONp (13) and
2-NHOHNp (14), see Table 1.
Interestingly, in the case of the 40-tert-butyl sub-
stituted compounds 8 and 9, the mutagenicity is
higher in the absence of S9-mix. Without S9-mix,
the dose-response curve of the hydroxylamine 9
shows an almost linear increase, indicating that no
cell toxicity influences the measurement up to a dose
of 500 ␮g/50 ␮l. In contrast to the non-alkylated
biphenyls 2 and 3, the presence of S9-mix reduces
the cytotoxic effect. The nitro compounds 4 and 7 do
not show any cell toxicity, either.
4. Discussion
4.1. The reduction steps from the nitro group to the
hydroxylamine
In all cases, the hydroxylamines and the nitroso
compounds are mutagenic even at low concentra-
tions, but the mutagenicity is seriously influenced by
cytotoxicity already at moderate doses. The addition
of metabolically activating S9-mix seems to have a
The dose-response curves of the hydroxylamines
3, 6, 9, 12 and 14 and the corresponding nitroso com-
pounds 2, 5, 8, 11 and 13 are very similar to each
other, whereas they are rather different from that of the

188
T. Haack et al. / Mutation Research 491 (2001) 183–193
Table 1
Mutagenicity of the non-alkylated aromatic hydroxylamines 3, 12, 14 and the corresponding nitroso compounds 2, 11 and 13
Compound
Dose (␮g/plate)
Revertants
TA98
TA100
−S9
+S9
−S9
+S9
1a
4-NBp
20
100
500
96
233
290
37
2149b
288
629
882
849
0 (tox)
32
3136b
249
516
513
131
406
508
67
1151c
304
347
795
885
207
902d
522
502
387
305
488
1285
1294
206
795c
716
1086
1003
425
363
146
2351c
769
1112
1005
531
51
146
2351c
520
DMSO
Positive control
4-NHOHBp
3
10
20
50
100
500
674
1134
1176
506
43
1628c
340
0 (tox)
DMSO
Positive control
4-NOBp
158
895d
557
311
272
167
2
10
20
50
100
500
778
1111
1104
614
43
1628c
809
1244
2196
38
1428c
1695
1652
888
347
0 (tox)
32
3136b
615
1512
1605
33
1794b
225
0 (tox)
DMSO
Positive control
4-NF
158
895d
365
834
0
134
836d
140
17
10a
12
10
20
50
867
1840
114
1774c
613
462
297
THF
Positive control
2-NHOHF
10
20
50
122
38
0 (tox)
100
500
0 (tox)
0 (tox)
25
239
0 (tox)
0 (tox)
0 (tox)
160
1266d
221
0 (tox)
0 (tox)
164
2285c
552
562
596
0 (tox)
0 (tox)
164
2285c
503
899
791
0 (tox)
0 (tox)
164
2285c
1269
1017
371
0 (tox)
0 (tox)
164
2285c
DMSO
39
2171c
2116
1363
959
798
0 (tox)
39
2171c
209
472
474
708
0 (tox)
39
2171c
218
481
719
Positive control
2-NOF
3474b
329
11
14
13
10
20
50
100
500
355
122
0 (tox)
0 (tox)
0 (tox)
0 (tox)
0 (tox)
0 (tox)
25
3474b
257
473
685
111
0 (tox)
25
3474b
569
896
194
DMSO
Positive control
2-NHOHNp
160
1266d
792
1198
1378
1192
10
20
50
100
500
0 (tox)
DMSO
Positive control
2-NONp
160
1266d
746
1168
1340
893
0 (tox)
160
1266d
10
20
50
100
500
0 (tox)
0 (tox)
25
923
0 (tox)
39
2171c
DMSO
Positive control
3474b
a Mutagenicity data taken from [3].
b 1-Nitropyrene, 2.5 mg.
c 2-Aminoanthracene, 2.5 mg.
d Sodium azide, 5 mg.

T. Haack et al. / Mutation Research 491 (2001) 183–193
189
Fig. 2. Comparison of the dose-response curves of 4-nitrobiphenyl (1), 4-nitrosobiphenyl (2) and 4-hydroxylaminobiphenyl (3) in TA100.
The tests were carried using the Ames-test [8] and the Salmonella typhimurium tester strains TA98 and TA100.
respective nitro compounds 1, 4, 7 and 10. This effect
was found in both tester strains, TA98 and TA100, and
was shown to be independent of the addition of rat
liver S9-mix, see Tables 1 and 2, and Figs. 1–5. Bulky
alkyl groups far away from, or near to, the functional
group NO2, which we previously showed to carry out
a remarkable influence on the mutagenic properties
of aromatic nitro compounds [2,3], do not disturb that
effect. The early decrease of mutagenicity induced by
cell toxicity in the dose-response curves of the hydrox-
ylamines 3 and 12 and the nitroso compounds 2 and
11, compared to their corresponding nitro compounds
1 and 10, shows that the reduction of the nitro group
to the nitroso functionality is rather slow. Thus, the
metabolically activated reduction procedure of nitro
compounds obviously consists of a “difficult” reduc-
tion from the nitro to the nitroso species, followed
by a more “easier” reduction from the nitroso to the
hydroxylamine function. Interestingly, a similar ob-
servation has earlier been made in the 1-nitropyrene/
1-nitrosopyrene/1-hydroxylaminopyrene system by
Heflich et al. [11]. They found that 1-nitrosopyrene
is at least 20-fold more mutagenic than 1-nitropyrene
in Salmonella typhimurium TA1538, with the same
Fig. 3. Comparison of the dose-response curves of 2-nitrofluorene (10), 2-nitrosofluorene (11) and 2-hydroxlaminofluorene (12). The tests
were carried using the Ames-test [8] and the Salmonella typhimurium tester strains TA98 and TA100.

190
T. Haack et al. / Mutation Research 491 (2001) 183–193
Table 2
Mutagenicity according to the Ames assay [8] in Salmonella typhimurium TA98 and TA100 of the alkylated aromatic hydroxylamines 6
and 9 and the nitroso compounds 5 and 8a
Compound
Dose (␮g/plate)
Revertants
TA98
TA100
−S9
+S9
−S9
+S9
4b
3-tBu-4-NBp
20
100
500
31
33
39
44
43
43
117
124
139
131
129
188
DMSO
Positive control
3-tBu-4-NHOHBp
30
45
116
117
830c
132
375
640
780
442
25
998d
107
358
772
1097
883
39
864e
824
1033d
517
6
10
20
50
100
500
1399
1729
1825
1032
160
1266e
679
1202
1586
2360
2050
160
1266e
206
1315
1806
1783
1290
164
2285d
489
1022
1745
2099
1261
164
2285d
209
DMSO
Positive control
3-tBu-4-NOBp
3464c
134
285
439
785
1146
25
3474c
45
53
2171d
95
5
10
20
50
100
500
283
541
1162
1829
39
2171d
62
DMSO
Positive control
40-tBu-4-NBp
7b
20
100
500
63
68
203
210
209
227
50
DMSO
Positive control
40-tBu-4-NHOHBp
37
67
207
206
2149c
34
1151d
57
902e
167
795d
277
9
10
20
35
65
191
360
50
55
73
218
352
100
500
92
108
205
39
258
746
160
1266e
220
368
524
164
2285d
314
438
25
3474c
42
DMSO
Positive control
40-tBu-4-NOBp
2171d
50
8
10
20
73
62
320
380
50
86
56
407
446
100
500
154
281
25
3474c
67
1061
1707
160
1266e
546
978
164
2285d
160
39
2171d
DMSO
Positive control
a For comparison, the nitro compounds 4 and 7 are also added [2,3].
b Mutagenicity data taken from [2] and [3].
c 1-Nitropyrene, 2.5 mg.
d 2-Aminoanthracene, 2.5 mg.
e Sodium azide, 5 mg.

T. Haack et al. / Mutation Research 491 (2001) 183–193
191
Fig. 4. Comparison of the dose-response curves in TA98 of 3-tbutyl-4-hydroxylaminobiphenyl (6), with its nitroso (5) and nitro (4)
analogues. The tests were carried using the Ames-test [8] and the Salmonella typhimurium tester strains TA98 and TA100.
deoxyguanosine-adduct being formed as from the cor-
responding hydroxylamine. These authors proposed
the nitroreductase, which is responsible for the ini-
tial reduction of 1-nitropyrene, to be the rate-limiting
factor. Howard et al. found that 1-nitropyrene is
rapidly reduced to 1-aminopyrene by the strains TA98
and TA100 of Salmonella typhimurium, confirming
that these tester strains are able to reduce aromatic
nitro and nitroso compounds without metabolically
activating S9-mix [12]. The ability to reduce these
functional groups is dependent on the size of the
aromatic system.
We have reported before that a bulky alkyl group
close to the functional group of 4-NBp (10) disturbs
the enzymatic reduction from the nitro to the nitroso
compound, making the substrate non-mutagenic [2].
This is either due to a sterical inhibition of the nitrore-
ductase by the bulky group, or it might be caused by an
“out of plane” orientation of the nitro group with re-
spect to the ring planarity [2]. However, the reduction
of the nitroso compound 5 to the directly mutagenic
hydroxylamine 6 is not at all influenced by the bulky
group, and S9-mix does not change the dose-response
curves, see Fig. 3. This means that the second
Fig. 5. Comparison of the dose-response curves in TA98 of 40-tbutyl-4-hydroxylaminobiphenyl (9) with its nitroso (8) and nitro (7)
analogues. The tests were carried using the Ames-test [8] and the Salmonella typhimurium tester strains TA98 and TA100.

192
T. Haack et al. / Mutation Research 491 (2001) 183–193
reduction step from the nitroso to the hydroxylamine
functionality might be rather induced by some smaller
non-enzymatic substance than by the interaction with
a “larger” enzyme. This seems also to hold in the case
of other nitro, nitroso and hydroxylamine compounds,
see Table 1 and Fig. 3. Mutagenicity tests carried out
by Wirth et al. support that hypothesis. These authors
did not find a significant difference in the mutagenicity
of 2-NOF (11) in either nitroreductase proficient TA98
nitroreductase deficient TA98FR [13].
difference to DNA which has formed an adduct with
the non-alkylated biphenyl 3. Thus, an exact expla-
nation for the suppression of the mutagenicity of
40-tBu-4-NBp (7) cannot be given as yet. The anal-
ysis of reactions between the hydroxylamines 3 and
9, respectively, and DNA, or Salmonella bacteria,
might give some additional information. Appropriate
in vitro experiments are under investigation.
4.3. Substitution of hydroxylamines by nitroso
compounds in the Ames assay
4.2. The influence of a bulky alkyl group in
40-position of nitrobiphenyl (1)
Aromatic hydroxylamines are interesting substrates
in the Ames assay, because they give important
information about the mutagenic properties of in-
termediates arising from aromatic amines and nitro
compounds after metabolic activation in vivo. Nev-
ertheless, in most cases their preparation and their
purification is not easy because aromatic hydroxy-
lamines easily undergo disproportionation yielding the
corresponding amine and the azoxy compound [14].
Nitrosoarenes can more easily be prepared from the
amines by oxidation using a solution of Mo[O2]2O·
HMPT·H2O in 30% H2O2 [15]. They are stable up
to room temperature and can be purified using silica
gel chromatography. Since their dose-response curves
are very similar to that of the hydroxylamines with-
out any exception, see compounds 2 and 3, 5 and 6,
8 and 9, 11 and 12 in Figs. 2–5, the investigation
of the nitroso compounds in the Ames assay might
be worthwhile in order to get information about the
corresponding hydroxylamine, if the latter is difficult
or impossible to obtain.
In conclusion, we showed that the aromatic hydro-
xylamines 3, 6, 9, 12, 14 and their corresponding ni-
troso compounds 2, 5, 8, 11, 13 yield almost identical
results in the Ames assay using TA98 and TA100
without any exception. Even bulky alkyl substitution
(tert-butyl group) does not change that results, al-
though it was previously shown that alkyl substitution
has a remarkable influence on the mutagenicity of
an aromatic nitro compound [2,3]. Thus, when a hy-
droxylamine is difficult to prepare, the corresponding
nitroso compound might give a hint about the muta-
genicity of the hydroxylamine. Furthermore, it was
found that bulky alkyl groups like the tert-butyl group
can interfere with the mutagenicity of 4-NBp (1) in
at least two different ways, depending on the location
40-tert-Butyl-4-nitrosobiphenyl (8) and 40-tert-
butyl-4-hydroxylaminobiphenyl (9) are much less mu-
tagenic in TA98 and TA100 than their non-alkylated
parent compounds 2 and 3, see Fig. 5, although the
sterical influence of the tert-butyl group is located far
away from the functional group. Even in the absence
of S9, 9 shows some mutagenic activity at higher
concentrations. The loss of cell toxicity in the case
of 9 might indicate a decreased effective dose of the
substrate 9 in the cells. The interaction between the
substrates and the cells could in some way be dis-
turbed by the bulky substituent, probably during the
penetration of the compound through the cell wall. As
a consequence, the hydroxylamine 9 is degradated be-
fore larger amounts are able to reach the cell. Larger
amounts of the hydroxylamine 9 within the cell would
be likely to carry out at least some cytotoxic effect
at higher concentrations. Addition of S9-mix de-
creased the mutagenicity because the hydroxylamine
seems to be more quickly degraded to non-mutagenic
by-products. What are the reasons for the 40-alkylated
nitro compound 7 to be non-mutagenic at all, see
Fig. 5? First, the enzymatic reduction step from
40-tBu-4-NBp (7) to 40-tBu-4-NOBp (8) seems to be
affected by the alkyl substituent, because 7 does not
show any mutagenicity at all, even not on addition of
S9-mix, whereas the hydroxylamine 9 and the nitroso
analogue 8 both show at least some mutagenicity.
Secondly, most of the hydroxylamine 9 seems to be
degraded before it can pass the cell wall and form
adducts with DNA. Besides that, there is the possi-
bility that the attack of the hydroxylamine 9 on the
DNA is sterically disturbed, or that DNA modified
with 9 is harmless, because of some conformational

T. Haack et al. / Mutation Research 491 (2001) 183–193
193
of that group in relation to the functional group. A
bulky group close to the functionality (ortho-position)
appears to disturb enzymatic reduction. When the
alkyl substituent is located far away from the func-
tional group, e.g. in the 40-position of 4-nitrobiphenyl
(7), the influence is not yet understood. However,
our results suggest that a decrease in the effective
concentration of the test compound, or a conforma-
tional difference of the adduct as compared to the
non-alkylated adduct, might be responsible.
[6] S. Ning, X. Xiaobai, Reductive metabolism of 4-nitroso-
biphenyl by rat liver fraction, Carcinogenesis 118 (1997)
1233–1240 and earlier references cited.
[7] J. McCann, E. Choi, E. Yamasaki, B.N. Ames, Detection of
carcinogens as mutagens in the Salmonella/microsome test:
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(1975) 5135–5139.
[8] D.M. Maron, B.N. Ames, Revised methods for the Salmonella
mutagenicity test, Mutat. Res. 113 (1983) 173–215.
[9] T.B. Patrick, J.A. Schield, D.G. Kirchner, Synthesis of
fluoroaromatic amines, J. Org. Chem. 39 (12) (1974) 1758–
1761.
[10] E. Brill, The oxidation of some carcinogenic aryl-
hydroxylamines to nitroso derivatives with manganese
dioxide, Experientia 30 (1974) 835.
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