Reports in the literature on the breakdown pathways of phenolic antioxidants
and interactions of their metabolites with foodstuff components are scarce.
We previously reported that butylated hydroxyanisole (3-BHA; E320; CAS No.25013-16-5),
used widely to prevent oxidative degradation in oils, fats, and shortenings,
and 2-tert-butyl-hydroxyquinone (t-BHQ) are readily oxidized
to 2-tert-butylquinone (t-BuQ). The latter reacts with amines
to form red addition products (1). Recently Soma and Soma reported
on the reaction of anilines with the quinone ring of humate by Michael addition,
followed by oxidation to form stable amino-substituted quinones (2).
By way of multiple transformations, for example, in the biosphere or in
the diet, new, more hazardous substances may be formed. Combination effects
are therefore of particular practical interest; however, investigation of
this problem has been limited.
A wide variety of primary amines is found in foods, particularly foods
that are produced by or subject to microbial fermentation, such as cheese,
meat, and fish (3). The only two arylamines that seem to be widespread
are aniline and N-methylaniline. According to Neurath (4),
aniline is found primarily in rapeseed, a source of edible oil and protein
(120 ppm) and carrots (31 ppm), whereas N-methylaniline is found
in cheese (38 ppm). Other sources are given by Shephard et al. (5).
In addition, substituted anilines and halogenated derivatives, which are
intermediates in the environmental degradation of many pesticides and other
industrial chemicals, have different migration capacities, toxicities, and
other properties. The ability of such pollutants to enter and accumulate
in tissues may contaminate the entire food chain (6,7). Furthermore,
field observations and experiments showed that plants readily take up aniline
residues derived from phenylamide herbicides through their root systems
and translocate it (8). Another source of contamination is water,
because large quantities may be used to wash, heat (direct steam injection),
or reconstitute foods. Unfortunately, assuring that the water is not contaminated
via industrial pollution does not guarantee the absence of petrochemicals
in marine animals (9). Other aquatic organisms, either grown in the
water or serving as food for other marine animals, are frequently the source
of anilines (10).
Due to the complex nature of food, the analysis of potential contamination
or irreversible fixation of xenobiotics to food constituents is a massive
task. Thus, model reactions are frequently used and have contributed much
to the understanding of interactions of organic pollutants with food constituents.
For our studies we chose the substances BHA, its accessible metabolites
t-butylquinone and t-butylhydroquinone, and the arylamines
aniline and N-methylaniline as representative model substances. The
present study was undertaken to identify the reaction products and to assess
their potential to act as mutagens in the Salmonella typhimurium
assay (11). This was a first step in elucidating the biological and
toxicological properties of these new products.
Aniline and N-methylaniline (NMA; Merck, Darmstadt, Germany) were
used only after redistillation. 15N-enriched aniline-hydrochloride
(96.0%) was purchased from VEB-Berlin-Chemie and used without further purification.
The commercial preparation of BHA is a mixture of two isomers. The major
isomer is 3-t-butyl-4-hydroxyanisole (BHA) and the minor one is 2-t-butyl-4-hydroxyanisole.
BHA was obtained in >99% purity by fractional crystallization of the
commercial sample (Carl Roth, Karlsruhe, Germany) from hot hexane containing
3% acetone. t-BHQ was obtained from Fluka (Neu-Ulm, Germany); t-BuQ
was synthesized via oxidation of BHA as described previously (1).
For preparative thin-layer chromatography (TLC), silica gel-60 F-254
plates (thickness 2, 0.25 mm; Merck, Darmstadt, Germany) were used. All
other chemicals were used in the highest purity commercially available.
Beef extract and agar used to culture microorganisms in the S. typhimurium/
mutagenicity assay (11) were purchased from Difco Laboratory, Detroit,
Michigan. Detailed preparation procedures and positive control experiments
are described elsewhere (12).
Melting points, measured with a Büchi melting-point apparatus, were
left uncorrected. We determined the UV-VIS spectra using a Perkin-Elmer
Lambda 2 spectrophotometer. Infrared spectra were recorded on a Perkin-Elmer
Infrared spectrophotometer model 881 in KBr. The NMR spectra were measured
using Bruker AC-200 and AM-400 instruments in CDCl3. Mass spectra
were obtained on a Finnigan MAT 4000 using 70 eV ionizing electrons.
Preparation of Arylamine Adducts of BHA and Derivatives
We dissolved aniline (5 mM, 93.12 mg) and t-BuQ (0.5 mM, 82.10
mg) in 30 ml ethanol and allowed it to stand for 15 hr at 21°C
in the dark. The reaction mixture was extracted twice with 20 ml chloroform.
The combined organic fraction was treated with anhydrous sodium sulfate
and, after filtration and concentration, subjected to preparative TLC (thickness
2.0 mm), using light petroleum and acetone 8:2 v/v as eluent. Two red zones
were observed on the TLC plate. Each of them was extracted with chloroform
and rechromatographed on silica-gel layers (0.25 mm). We removed the red
zones with Rf 0.47 (major; I) and 0.55 (II), respectively,
and eluted them with 20 ml acetone. The eluates were then evaporated to
dryness on a rotary evaporator under reduced pressure to give red powders,
which were recrystallized from hot methanol/water. The crystalline red solids
were dried in a vacuum desiccator. Their melting points were 133-134°C
for I and 101-102°C for II.
To support our structure assignments, we also carried out the above synthesis
using 15N-aniline. The reaction products were separated and collected
in the same manner as described above. Recrystallized and vacuum dried material
was investigated by mass spectometry.
The reaction between NMA (5 mM, 107.16 mg) and t-BuQ (0.5 mM,
82.10 mg) was carried out as described above. Two red zones with Rf
0.39 (major, III) and 0.52 (IV), respectively, were eluted with 20 ml acetone
and evaporated to dryness on a rotary evaporator under reduced pressure.
The products were crystallized from hot methanol/water to give red solids.
We then analyzed the vacuum-dried products using the methods described above.
Their melting points were 88°C for III and IV 119°C
for IV. None of the compounds I-IV were found reactive toward nitrite in
high excess and at low pH.
Structural Determination of the Main Reaction Products
Compound I. Mass spectral and elemental analyses indicated the
molecular formula C16H17NO2. Analysis found:
C, 74.98%; H, 6.50%; N, 5.40%. Calculated: C, 75.27%; H, 6.71%; N, 5.49%.
The mass spectrum (Fig. 1) showed a molecular ion peak M+ at
m/z 255, a strong fragment at 212 (M-C3H7), 240 (M-CH3),
77 (C6H5+) and other low-mass ion peaks;
max(EtOH) 262 and 499 nm (18053 and 4233). The UV spectrum
showed a strong bathochromic shift on addition of alkali (pH > 8, 499-542
nm) suggesting the presence of an N-H group in ortho position to
a C=O group of the quinone ring. The most characteristic IR (KBr) absorption
bands appeared at 3250 cm-1 (NH-stretching), 2950 cm-1
(CH3-stretching and 1650 cm-1 (C=O stretching), due
to the quinoid ring.
The 15N-labeled compound followed the same general pattern
as set out above and showed a molecular ion M+ at m/z 256 and
a major fragment ion at m/z 213, supporting the presence of a single N atom
in this adduct. Further evidence for the structural characteristics was
obtained from 1H-NMR spectroscopy. One t-butyl group was
observed at 1.31 (s, 9H) and a typical set of two doublets at 6.52 (d, 1H)
and at 6.12 (d, 1H), both I = 2.4 Hz. With 15N the signal of
the proton in position to the Ph-15NH group was split into the
two doublets at 6.12 (dd, 1H, I1 = 2.4 Hz; I2 = 1.0
Hz). Five aromatic protons and an NH were observed in the range 7.6-7.4
(m, 5H) and NH (d, 1H, I = 92.5 Hz). All of these data indicate the structure
of the main product as that shown in Figure 1.
![](kalusfig1.GIF)
Figure 1. Mass spectra of the quinone adducts
isolated from reaction of BHA with aniline, 15N-aniline and N-methylaniline
as described in Materials and Methods.
Compound II. The purified minor product (II) also yielded the
same molecular formula, C16H17NO2, as compound
I. Its mass spectra showed M+ at m/z 255 and a strong fragment
at m/z 212;
max (EtOH) 262 and 495 nm (* 19545 and 3409).
The addition of alkali (pH > 8) also resulted in a bathochromic shift
from 498 to 510 nm. The NMR data show one t-butyl group at 1.25 (s,
9H) and a typical set of two singlets at 6.48 (s, 1H) and 6.02 (s, 1H).
The five aromatic protons and an NH were observed in the range 7.6-6.9 (m,
5H) and at 7.5 (s, 1H). The IR spectrum was similar to that of compound
(I). All these findings indicate the structure of II to be the isomeric
anilinoquinone shown in Figure 2.
![kalusfig2](kalusfig2.GIF)
Figure 2. Possible mechanism for the formation
of quinone adducts initiated by interactions of BHA with aniline (I, II;
R=H) and N-methylaniline (III, IV; R=Me).
![](kalustab1.GIF)
Compound III. The major product obtained from the reaction of
t-BuQ with N-methylaniline gave a molecular formula of C17H19NO2,
which was supported by its mass spectrum (Figure 1), giving M+
at m/z 269 and strong fragment ions at m/z 254 (M-CH3); 226 (M-C3H7);
77 (C6H7+). Analysis found:
C, 76.00%; H, 7.13%; N, 5.20%. Calculated: C, 76.10%; H, 7.11%; N, 5.20%;
*max (EtOH) 475 and 257 nm (16809 and 3822). Its IR spectrum
showed the loss of the NH absorbance at 3250 cm-1. Further characteristic
bands appeared at 2950 cm-1 (asymmetric methyl stretching vibration),
2870 cm-1 (symmetric stretching), 1260 and 1210 cm-1 (tertiary
butyl vibration). The NMR data show the t-butyl group at 1.01 (s,
9H) and methyl protons at 3.21 (s, 3H). A typical set of doublets at 5.79
(d, 1H; I = 2.8 Hz) and 6.41 (d, 1H; I = 2.5 Hz) together with the aromatic
protons in the range at 6.88- 7.33 (m, 5H) were good evidence for the assigned
structure (III; Figure 1).
Compound IV. The mass spectrum produced the same characteristic
fragmentations as shown in the assigned structure (III); *max(EtOH)
254 (shoulder) and 492 nm (3040 sh). The NMR data [*: 1.24 (s, 9H), 3.25,
(s, 3H), 5.70 (s, 1H), 6.27 (s, 1H), 6.90-7.39 (m, 5H)] indicate the structure
of the compound (IV; Fig. 2).
Reaction Yields and Mutagenicity
After completion of the structural identification of the major products,
we attempted to determine their yields in reactions run at various physiological
pH values. Observations during workup indicated the rates of appearance
of some of the products depended on pH. In general, the initial reaction
conditions and separations were as already described. The substances were
removed from the TLC plates, dissolved in chloroform, evaporated to dryness
,and the residues dissolved in ethanol for UV spectrophotometric analysis.
The yields are shown in Table 1.
Mutagenicity was assayed according to the original protocol of Ames et
al. (11). We used the histidine auxotrophic strains TA98 and TA100
of S. typhimurium as test organisms. Tester strains were maintained
and checked for retention of properties as recommended by Maron and Ames
(13). We prepared hepatic post-mitochondrial fractions (S9) from
three pooled livers of male Sprague-Dawley rats treated with Aroclor-1254.
The S9-fraction was used at a concentration of 0.1 ml/ml of S9-mix. Test
samples were dissolved in dimethyl sulfoxide. All the experiments were performed
in two separate assays with triplicate plates.
N-methylaniline caused no increase in the number of revertants
per plate in S. typhimurium strains TA98 and TA100 at concentrations up
to 2.0 mg/plate with or without S9 mix. Because of the low yield of compound
IV, insufficient material was available to conduct testing.
The fate of available aniline residues in food constituents is difficult
to characterize because, in general, foodstuffs are so heterogeneous and
complex. However, the chemical bonding of aniline to food additives can
be characterized through models that allow the determination of suspicious
products. In this report we examined the chemical reactions initiated by
the food additive BHA and its main metabolites with some xenobiotic food
constituents like aniline and NMA, which are also known to be breakdown
products of some pesticides.
Under the mild reaction conditions described, both aniline and NMA yielded
red products with t-BuQ. No visible change was observed in individual
solutions of the two phenolic antioxidants BHA and t-BHQ in aniline
or NMA during the same time period. The results of the investigations are
summarized in Table 1. The maximum yield of the anilinoquinones depended
on the combination of concentrations of the substrates and on the initial
pH value.
Based on our model experiments with 15N-labeled aniline, we
suggest that the reaction proceeds basically as in Figure 2. This type of
mechanism is often postulated to explain the binding of substituted anilines
to phenolic humus constituents by way of enzymatic oxidation (14,15).
Thus it seems likely that the phenols will be first oxidized by oxidants
like O2 and MnO2 (pyrolusite) or the food additives
NaNO2 and KIO3 to the corresponding quinone (16).
The oxidation reactions were also observed by incubating BHA with liver
microsomes (17). The quinone is an obvious oxidation product of both
BHA and t-BHQ. Recent work by Bergmann et al. has indicated the participation
of the t-butylsemiquinone anion radical during the microsomal incubation
of t-BuQ and t-BHQ (18). The studies described above,
as well as those cited by Warner et al.(19) demonstrate that phenolic
antioxidants undergo a sequence of reactions in food. In a subsequent step
the attack of the nucleophilic amine occurs at the less sterically hindered
ring position of the quinone. It was therefore not unexpected that only
one additional isomeric product was observed and in low yields.
It is generally accepted that univalent reduction of quinones is a major
cause of their intrinsic toxicity (20). Data on the toxicology of
t-BuQ is not available. However, a mutagenic compound arising from
BHA upon treatment with nitrite under acidic conditions has been identified
as t-BuQ (21). In recent studies, no mutagenicity was detected
in the ROS-sensitive tester strains S. typhimurium TA102 and TA104
(22). Our results, summarized in Table 2, demonstrate that t-BuQ
showed no mutagenic response at two nontoxic doses in the standard Salmonella/microsome
test. With increasing concentration of the quinone a cytotoxic effect was
observed, apparent in the decreasing yield of macroscopically visible His+
revertant colonies and in the thinning of the background lawn. This observation
is in agreement with the investigations of Kahl et al. (23); It was,
moreover, partly reduced by the addition of S9-mix. The thinning of the
background lawn was not quantitatively determined, hence we may make no
statement concerning the proportionality to the His+ revertants.
We are looking into potential antigenotoxic effects using eukaryotic systems
and will report these studies later. The toxicity was completely abolished
in the adduct (I) prepared by the reaction of t-BuQ with aniline.
In contrast to compound I, the isomeric compound II was cytotoxic at high
concentrations. The main product in the reaction of t-BuQ with NMA
(III) was cytotoxic in the S. typhimurium assay. All of the investigated
products failed to induce any increases in revertant numbers in either of
the strains, with or without S9-mix.
![](kalustab2.GIF)
N-nitroso-N-methylaniline is a potent esophageal carcinogen
(24). Spinach and many pickled vegetables (cucumber, celery, and
others) are known to be sources of one of its precursors, the NMA used in
this study. From our work it seems possible that BHA and its quinoid metabolites
could exert a protective effect against the formation of N-nitroso-N-methylaniline.
This might involve diversion of both the aniline and nitrite to form the
innocuous nitrite-inactive compounds found in this study. This conclusion
is important in connection with Kato's recent observation of the transformation
of arylamines into direct-acting mutagens by the action of nitrite (25).
Furthermore, a diminution in the superoxide anion (18) may occur
as the metabolites are transformed into anilino adducts. Clearly, the availability
of these reagents and the relative kinetics of their reactions with each
other ("additive cocktail") will determine the importance of these
effects. Delimitation of the conditions favoring protection in such cases
will be the subject of further work.