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Contribution of primary aromatic amines to the mutagenicity of gasifier tars and coal oils
Mutation Elsevier
Research, 155 (1985) l-16
MTR 00939
Contribution
of primary
W.E. Bechtold,
aromatic amines to the mutagenicity and coal oils J.S. Dutcher,
of gasifier tars
A.L. Brooks and R.F. Henderson
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Albuquerque, NM 87185 (U.S.A.)
Research Institute, P.O. Box 5890,
(Received 10 December 1983) (Revision received 14 May 1984) (Accepted 14 September 1984)
Summary Two reactions that chemically alter primary aromatic amines (PAA) were used to assess the contribution of these compounds to the indirect bacterial mutagenicity of tar from an experimental low Btu gasifier. The first reaction, nitrosation, effectively eliminated the mutagenicity of several PAA standards and a coal oil when run in a low pH media (1.2). When applied to gasifier tar, extensive direct (not requiring metabolic activity) mutagenicity was generated. This direct mutagenicity limited the interpretation of results. When the pH of the reaction media was raised to 2.5, the mutagenicity of PA.4 standards and the coal oil were still greater than 90% eliminated, however, no direct mutagenicity was observed for the gasifier tar. Furthermore, only 61% of the indirect (requiring metabolic activation) mutagenicity was eliminated. Acetylation reduced the indirect activity of most primary amine standards by greater than 79%. Acetylation of the tar likewise eliminated part, but not all, of the activity, whereas most of the activity of the coal oil was eliminated. These results indicated that a much lower percentage of the mutagenic activity of low Btu coal tar samples was due to primary aromatic amines than was the case for coal oil.
Primary aromatic amines (PAA) are believed to contribute significantly to the observed bacterial mutagenicity and potential hazards associated with coal-derived synthetic fuels (Pelroy and Stewart, 1982). In principle, the relative contributions of PAA to the overall observed mutagenicity of complex mixtures could be determined by isolation of pure PAA fractions. Unfortunately, the careful and extensive separations necessary to isolate such pure PAA fractions have proved difficult and time-consuming because of their low concentrations in the complex mixtures and high chemical reactivities (Later et al., 1982; Ton&ins and Ho, 1982; Haugen et al., 1981b). Other means of assessing the contributions of PAA to the bacterial mutagenicity have therefore been sought. 0165-1218/85/$03.30.0
1985 Elsevier Science Publishers
Reaction with nitrous acid has been a successful technique for determining the contributions of PAA to the bacterial mutagenicity of process solvents (PS) and heavy distillates (HD) from solvent refined coal (SRC) (Pelroy and Stewart, 1982). Nitrous-acid-dependent decreases in indirect bacterial mutagenic activity (requiring rat-liver metabolic enzymes) were observed for numerous PAA standards, whereas mutagenic activity for non-PAA polycyclic aromatic hydrocarbons (PAH) and aza-arenes remained unchanged. The observation that more than 90% of the indirect activity for PS and HD disappears after reaction with nitrous acid led Pelroy and Stewart (1982) to conclude that PAA are quantitatively important in determining the mutagenicity of these mixtures. In
B.V. (Biomedical
Division)
8
their study, increases in direct mutagenicity accompanied decreases in indirect mutagenicity. Although slight in the case of PS and HD, side reactions creating direct-acting mutagens such as nitroso and nitro PAH may complicate the interpretation of the effect of nitric acid on other complex mixtures. Thus, Haugen et al. (1981a) developed methods for nitrous acid treatment specifically designed to minimize these side reactions. Under their conditions, generation of direct activity by reaction of nitrous acid with benzo[alpyrene (BaP) was shown to be minimal, whereas the indirect mutagenic activity of 2aminoanthracene was decreased to 5% of its original value. The contribution of PAA to the mutagenic activity of low Btu gasifier tars is also of interest because the basic fraction in which PAA would be found has high specific indirect mutagenic activity (Royer et al., 1983). We report two methods to assess the contribution of PAA to the mutagenicity of tar from an experimental low Btu gasifier. For validation of the methods, a coal oil similar in nature to SRC-II liquids was used for comparison. The first method, nitrous acid treatment, was shown to generate extensive direct activity in the gasifier tar using the conditions of Haugen et al. (1981a). At a higher pH, generation of direct activity was minimized, whereas indirect activity was significantly but not totally decreased. The second method, acetylation, also did not eliminate the indirect activity completely. These two methods indicated that PAAs account for only part of the observed mutagenic activity in low Btu coal tar.
(CRM) acquired from the Fossil Fuels Research Matrix Program (Oak Ridge National Laboratory). Methylene chloride, benzene, and dimethyl sulfoxide (DMSO) were purchased from Burdick and Jackson Laboratories. Pyrene, 2-aminoanthracene, 2-aminofluorene, 3-aminofluoranthene, laminopyrene, 6-aminochrysene, 9-aminophenanthrene, carbazole, and phenanthridine were obtained from Aldrich, 2-Aminofluorene, 6-aminochrysene, and l-aminopyrene in DMSO showed some direct bacterial mutagenic activity, and gas chromatography/mass spectroscopy (GC/ MS) showed contamination from the corresponding nitro derivatives. These three amines were therefore purified by preparative silica gel thin-layer chromatography (TLC). TLC plates were eluted with a 3 : 1 mixture of benzene: methylene chloride, scraped, and the silica gel extracted by sonication with methylene chloride. Purified compounds showed no traces of nitro derivatives by GC/MS analysis, and no direct mutagenic activity was detected. Mutagenesis assay Chemical standards, samples, and reaction products were tested for biological activity in Salmonella typhimurium strain TA98 as previously published (Ames et al., 1975). Each concentration was tested in triplicate with 5 concentrations per chemical. The mutagenic activities of the chemicals were evaluated both with (indirect) and without (direct) Aroclor-125Cinduced rat liver homogenate fraction (S9) as a source of metabolic enzymes. The number of revertant colonies was
\ /b
Materials and methods Materials Gasifier tars were collected from the tar trap of an experimental low Btu gasifier unit at the Morgantown Energy Technology Center, Morgantown, WV (METC). The coal gasifier was a stirred-bed unit using a modified Lurgi process. The gasifier and cleanup system have been described elsewhere (Newton, 1980). The tar trap was positioned in the process stream following a cyclone, which removed ash, and a humidifier which cooled and condensed the tars. Coal oil samples were comparative research materials
H,O
.N2@
Acetic NH2
Pg. 1. Reactions anhydride.
Anhydride of PAA with (A) nitrous
Y-KCH3 H 0 acid and (B) acetic
9
counted on an Artek Model 800 automatic colony counter and related to the concentration in the bacterial plate using a linear least squares regression analysis. The biological activity is thus reported as revertants/pg extract using the linear portion of the dose-response curve. The linear portion of the curve was defined in every case by a minimum of 3 points. Acceptability of points was based on arriving at a correlation coefficient of 0.95 in the regression analysis. Values used in direct comparisons were derived on the same days to minimize day-to-day variation in the tester strain. Nitrosation reactions Treatment of primary aromatic amines with nitrite in acid results in the formation of a diazonium salt, followed by decomposition to a phenol (Fig. 1). Initial nitrosation reaction conditions (method I) were essentially those of Haugen et al. (1981a), with only slight variations. About 5-10 mg of sample was dissolved in 0.5 ml of DMSO and added to a solution of NaNO, (5 mg/ml) in HCl (0.3%, pH 1.2). For the control, NaNO, was omitted. Reactions were allowed to proceed for 2 min at room temperature and were stopped by the addition of 1.5 ml of 1 N NaHCO,. The materials were lyophilized overnight and then dissolved (suspended) in DMSO for bacterials mutagenicity testing. Nitrosation reactions were also produced by methods similar to those of Tsuda et al. (1981) (method II). Typically, PAA or complex mixtures were dissolved in DMSO to concentrations of 1 mg/ml and 10 mg/ml respectively. Aliquots of 0.4 ml each of the DMSO mixtures were added to 3.2 ml of citrate/Na,HPO, buffer. Citrate (50 mM) and phosphate (50 mM) buffers were mixed to form solutions of pH 2.5 and 4.0. Nitrosation reactions were conducted by adding 0.4 ml sodium nitrite solution (10 mM). As controls, either 0.4 ml of buffer was added, or nitrite was added without mutagens. Reactions were terminated by the addition of 0.2 ml of 0.25 M sodium bicarbonate to 0.8 ml of the reaction mixtures after 0.5-5 h. All solutions were filter sterilized before use in the Ames test. Acetylation reactions Treatment of PAA with acetic anhydride
results
in the acetylation of primary amines to amides (Fig. 1). For acetylation, 10 mg of standard or complex mixture was dissolved in 4 ml of a 20 : 1 solution of methylene chloride in pyridine, and 50 ~1 of acetic anhydride was added. Again, as a control, acetic anhydride was omitted. Reactions were stopped after 6 h by the addition of 0.6 ml of 1 N NH,OH, followed by enough ethanol to produce a homogenous solution. The solvents were evaporated under a stream of nitrogen, and the ammonium acetate was removed by sublimation in vacua at room temperature. Results
Changes in mutagenicity by nitrosation (method I) As observed by Haugen et al. (1981a), nitrosation by method I was effective in reducing the indirect bacterial mutagenicity of several PAA standards to less than 10% of the unreacted value. Furthermore, nitrosation under these conditions reduced the indirect bacterial mutagenicity of the coal oil sample to less than 10% within 3 min of the onset of the reaction without the generation of direct mutagenicity. When applied to tar from the low Btu gasifier, no reduction in the indirect muta-
15
. i
l =with
S-9
A= without
S-9
I
!
I
I
I
2
4
6
8
10
TIME (mid Fig. 2. Kinetics of reaction of gasifier tar trap tar with nitrous acid (method I). Conditions: n, with S9; A, without S9.
10
genie activity was noted (Fig. 2). This may be due to the simultaneous generation of direct mutagenic activity. In fact, within 20 set of the onset of the
reaction almost 2 revertants/pg of direct activity were generated, and direct activity continued to rise with time, as did the indirect activity.
TABLE 1 TA98 MUTAGENICITY OF 2-AMINOANTHRACENE (2AA) AND GASIFIER TAR TRAP TAR (REVERTANTS/ng) FUNCTION OF pH AND TIME OF NITROUS ACID TREATMENT (METHOD II) Time (h)
0.0
a
Sample
2AA
Amount/
pHb
plate (ng)
2.5
0.0 0.1 0.2 0.4 0.8 rev/ng
Tar 2.0 4.0 8.0 16.0 rev/ng 0.5
2AA
0.0 0.1 0.2 0.4 0.8 rev/P g
Tar
0.0 2.0 4.0 8.0 16.0 rev/P g
5.0
2AA
0.0 0.1 0.2 0.4 0.8 rev/pg
Tar
0.0 2.0 4.0 8.0 16.0 rev/pg
4.0
-s9
+s9
16 +3 26 *3 22 *2 18.2*2 21 56 l>
25 99 233 542 988 1271
0.0 17 21 22 20 l>
AS A
*2 +4 *3 *5
16 25 25 17 22 1>
*3 *1 *l *3 +3
16 18 29 31 37 l>
-13 f0 *1 *2 *7
16 18 18 21 22 l>
&3 rtl *O *o *3
16 *3 30 *2 48 *4 70 zt8 87 *l 3.8rtO.9
-s9 + 2 f 4 f 1 *51 k92’ *69
16 k3 110 *lo 179 *22 244 *20 339 *29 15.4+ 2.3 25 27 39 35 66 51
f 2 f 3 f 1 + 6 f lC *14
25 f 2 55 f 8 77 *18 96 +ll 133 +18” 5.3+ 0.6 25 23 38 44 55 40
+ 2 + 5 k10 +3 *14= *11
25 + 2 56 *16 78 f 9 110 *13 149 529” 6.4* 0.8
16+ 26+ 16& 17+ 16f 1>
+s9 3 7 3 1 6
25 f 2 22+ 3 13* 4 22k 8 35* 5 l>
25 97 267 977 1308 1763
16+ 59 f 109 f 186 f 349 f 20.5+
16+ 24k ll& 24+ 19* 12
3 1 2 1 1
25 98 269 773 1321 1762
16+ 14* 15+ 22+ 21+ 1>
3 3 2 3 8
25 91 170 216 292 13.0
16+ 3 19*11 22* 2 22+ 5 22*17 1>
25 69 131 393 932 1266
16& 22& 18+ 18* 29+ 12
3 2 9 1 2
f 2 f 5 f 11 + 6 + 61’ k413 325 + 11 1 13 53 0.4
f 2 f 1 f 28 f 77 k117’ 5173 f 2 + 11 f 16 k 8 f 18’ + 2.8 + k f f + f
2 17 54 30 57 68
25 f 2 58 f 6 80 + 6 196 +124 229 f 6 12.6+_ 3.7
’ Sample control - determined with buffer at pH 2.5 and 4.0, but without nitrite. b Determined without (- S9) and with (+ S9) Aroclor-1254-induced rat-liver microsomes (9000 X g fraction). ’ Significance of mean response established by detection of mutagenicity at greater than 2 times background.
2
11 TABLE 2 MUTAGENICITY OF STANDARDS NITROUS ACID = Chemical
Amount/ plate ( c1g)
2-Aminoanthracene
0
0.1 0.2 0.4 0.8 rev/pg 3-Aminofluoranthene
0 0.1 0.2 0.4 0.8 1.0 rev/pg
2-Aminofluorene
0 0.1 0.2 0.4 0.8 1.0 rev/p g
6-Aminochrysene
0 0.1 0.2 0.4 0.5 rev/yg
9-Aminophenanthrene
0 0.1 0.2 0.4 0.8 1.0 rev/p g
l-Aminopyrene
0 0.05 0.1 0.2 0.4 0.5 rev/y g
Carbazole
0 50 100 200
AND COMPLEX
MIXTURES
Control b (rev/pg) -s9
Reaction (rev/pg)
+s9
16+ 26+ 22* 18k 21+l>
3 3 2 2 6
17+ 28k 20* 31* 36+ 65k l>
2 3 7 4 3 6
19+ 23k 26k 18* 21* 29+ l>
3 7 2 5 7 1
24*11 21* 3 17* 4 16k 2 22* 9 12 19+ 16f 20+ 20f 26+ 27k l>
3 2 4 2 5 5
27k 20+ 24+ 24k 18k 23* l>
5 9 6 3 2 5
24k 30+ 24+ 32k 12
1 8 5 6
IN TA98 BEFORE AND AFTER TREATMENT
-s9
+s9
25 99 233 542 988 1271
+ 2 f 4 f 1 f 51 f 92’ f 69
16 25 25 17 22 l>
f f f + f
24 49 73 169 555 872 904
f 4 f 6 f 4 f 22 f 41 f 71’ *110
17 20 20 21 22 35 l>
+ f + + f f
32 + 3 49 f 4 62 f 7 91 f 9 153 f 8 208 f 34’ 171 f 12
19 23 19 26 30 30 l>
+3 f f f + *
f 1 f 8 f 12 k263 k166” f 774
24 27 19 21 20 l>
f + * f f
f f f f f f f
3 8 5 21 32 47’ 54
19 19 24 23 18 30 l>
+ f f + f f
39 f 2 38 + 9 52 f 14 66 f 2 120 f 10 150 f 13c 245 f 13
27 17 19 22 18 24 l>
f f f + + +
78 37 84 1474 1889 5094 32 38 55 114 421 518 570
28 33 35 33 l>
f f f f
1 4 16 6
24 25 54 113 l>
Remaining (%) d
3 1 1 3 3
2 5 1 4 4 7
4 3 2 1 3
1 4 2 3 3
3 6 7 5 1 3
5 1 8 5 2 5
f 11 +_ 4 f 13 + 7
25 27 39 35 66 51
f 2 f 3 + 1 zt 6 f lC * 14
24 23 26 35 33 31 l>
f 4 f 8 + 7 f 10 f 4 + 1
32 35 29 35 39 39 l>
+ f f f * f
28 35 3i 176 295 659
4
0 3 8 8 8 3 2 0 f 1 + 3 f 10 f 14 + 31’ k131
32 27 30 31 35 32 l>
f f + + * +
39 32 32 31 28 31 l>
f 2 f 1 f 9 + 6 + 10 f 6
28 37 51 79 l>
f f f f
13
3 1 2 7 9 5 0
0 1 3 6 5 0
WITH
12
TABLE
2 (continued)
Chemical
Amount/ plate (as)
Phenanthridine
Coal oil A
Tar trap tar
’ b ’ d
Control -s9
b (rev/p&
Reaction
+s9
0 2 4 8 16 20 rev/u g
24*11 21* 14* 17* 17+ 14+ l>
0 2 4 8 16 20 rev/ng
21* 25? 25+ 19* 21* 28+ I>
2 7 4 1 4 1
0 1 2 4 8 10 rev/pg
21* 16+ 24k 18* 22+ 19* 11
2 1 5 1 5 1
I 5 2 1 4
(rev/p@
-s9
28 + 1 24 rt -I 34 * I 54 * 9 155 * 11’ 152 + 21 9.5* 1.4
24 13 15 18 16 11 l>
32 + 47 k 83 k 149 * 324 + 198 ; 19.8k
4 6 14 15 40” 17 0.9
32 + 4 39 f 3 68 * 8 123 f 15 156 + 11 185 f 12’ 15.25 2.1
Remaining
(%) d
+s9 kll f f f k f
28 * 31 f 50 * 61 f 162 f 199 * 9.5*
1 1 17 26 23r 14 0.7
100
21 f 2 31 f 5 26 f 2 31 f 7 53 *lo 87 f 9’ 3.6* 0.9
32 f 42 f 35 f 40 * 72 f 61 + 1.7*
4 4 2 7 19 9’ 0.6
9
21 24 24 24 35 35 l>
32 * 38 + 32 f 63 f 78 ? 83 k 5.9?
4 1 4 6 6 3’ 1.6
39
_t + + + k *
5 I 7 3 9
2 4 3 8 6 8
Without (- S9) and with ( + S9) Aroclor-1254-induced rat-liver microsomes (9000 X g fraction). Control reactions have buffer but no sodium nitrite added. Significance of mean response established by detection of mutagenicity at greater than 2 times background. Ratio of mutagenicities before and after reaction, with S9.
Changes in mutagenicity by nitrosation (method II) Nitrosation reaction conditions were sought that would reduce the indirect mutagenic activity of PAA standards without generation of direct activity for tar trap tar. Time (0.5 and 5 h) and pH (2.5 and 4) were the two variables investigated. Results in Table 1 show that, regardless of the time of reaction, at pH 4, nitrous acid had limited effect in reducing the indirect activity of 2-aminoanthracene. At pH 2.5, 5 h of reaction time lowered the mutagenicity of 2-aminoanthracene to 2% of the unreacted standard. However, 3.8 revertants/pg of direct activity were also generated. The reaction time of 0.5 h at pH 2.5 proved optimum for reducing the indirect mutagenic activity of the aromatic amine standards without inducing direct mutagens. The indirect activity for the 2-aminoanthracene was reduced by 95%, but not direct activity was generated. Reaction conditions of pH 2.5 and 0.5 h were used to investigate the effect of
nitrous acid on several standards (Table 2). In all cases, nitrous acid reduced the mutagenicities of PAA standards to less than 16% of the control value. Mutagenicities for a secondary amine, carbazole, and an aza-arene, phenthridine, were not affected by nitrous acid treatment. When the coal oil sample was subjected to HCl without NaNO,, little change in the mutagenicity was noted (Table 2). However, addition of nitrite reduced the indirect activity to 9%, the value observed in HCl alone. Only a small increase in direct-acting mutagenicity was detected after treatment with nitrous acid. Treatment of tar trap tar with nitrous acid decreased but did not eliminate the indirect mutagenicity (Table 2). Thus, after treatment, 39% of the mutagenicity remained. The administration of HCl/NaNO1 had no effect on cell viability either in conjunction with PAA standards or with complex mixtures (Table 4). Viability studies were run
13 TABLE
3
MUTAGENICITY OF STANDARDS ACETIC ANHYDRIDE = Chemical
AND
Amount/
COMPLEX
Control
MIXTURES
b
IN TA98 BEFORE
Reaction
(rev/p@
AND
AFTER
Remaining
plate (PP) 2-Aminoanthracene
0.0 0.1 0.2 0.4 0.8 1.0 rev/y g
3-Aminofluoranthene
0.0 0.05 0.1 0.2 0.4 0.8 rev/pg
2-Aminofluorene
0.0 0.05 0.1 0.2 0.4 0.8 rev/pLg
6-Amincchrysene
0.0 0.1 0.2 0.4 0.8 1 .o rev/B g
9-Aminophenanthrene
0.0 0.025 0.05 0.1 0.2 0.4 rev/p g
I-Aminopyrene
0.0 0.025 0.05 0.10 0.20 0.40 rev/p g
1-Aminopyrene (repeat)
0.0 0.05 0.1 0.2 0.4 rev/p g
38 50 95 303 846 1246 1330
f 3 f 12 f 19 + 17 * 30 +261c +105
38 30 32 47 71 115 88
+ + f f * f f
3 6 7 18 19 30= 15
30 27 34 88 252 821 114
+ 8 k 3 * 10 * 1 f 17 +105’ k129
30 41 48 65 131 203 224
+ f k f + f k
8 10 4 14 12 19’ 15
20
30 27 47 72 110 246 284
f 8 + 12 * 4 + 5 f 9 + 8’ k 18
+ +
8 9 9 1 8 2’ 8
21
30 42 66 268 1570 175 2264
* 8 f 2 * 13 + 51 f 77c f 44 k462
30 41 38 42 75 164 374
f 8 f 6 + 3 f 9 * 12 + 22’ * 40
30 29 37 46f 59 105 196
+ f +
30 30 45 63 86 153
30 28 40* 42 47 78 59 30 25 31 33 43 179 133
+ f * f
TREATMENT
k 8 +_ 7 * 4 f 5 f 2 + 36’ + 61
30 30 30 27 21 30 12
k 8 + 14 f 10 k 2 f 3 5 6
8 2 5 6 + 5 + 6’ f 12
30 106 200 340 557 592 1254
* 8 * 15 * 10 f 19 + 24 +165’ *364
640
+ 8 f 3 + 4 f 10 f 7c f 21
30 179 320 488 635 1230
+ 8 f 13 f 30 + 40 f 4’ +254
804
(I%)
WITH
14
TABLE
3 (continued)
Chemical
Amount/
Control
h
31 23 24 31 12
* + k k
Reaction
(rev/p@
Remaining(W)
plate (p 9) Carbazole
0.0 25 50 100
rev/pg Phenanthridine
0.0 L
4 8 16 20 rev/p g Coal oil A
0 2 4 8 16 20 rev/p g
Tar trap tar
0.0 1
i 4 8 10 rev/p g
36 f 28 f 44 * 70 f 109 f 138 * 5.9*
5 2 3 6
31 26 30 38 1>
* * * f
5 1 6 2
1 9 4 3 10 20’ 0.2
36 f 1 32 f 6 42 + 5 59 f 8 110 f 19 120 * 19’ 5.1+ 0.3
86
28 f 1 30 f 2 61 * 8 79 * 15 189 + 7 212 * 22’ 10.4+ 0.8
28 f 1 27 f 10 30 k 6 46 + 13 70 * 3 82 * 7’ 3.1+ 0.1
30
31 * 42 k 61 * 120 f 131 * 162 + 12.2*
31 + 7 38 f 2 42 + 2 62 f 11 89 f 10 89 k 11’ 6.3* 0.8
52
7 6 5 4 5 17’ 2.8
a With Aroclor-1254-induced rat-liver microsomes (9000 X g fraction). b Control reaction, with methylene chloride and pyridine, without acetic anhydride. ’ Significance of mean response established by detection of mutagenicity at greater
simultaneously with mutagenicity and used the same mixtures at the same concentrations. Furthermore, controls using nitrite but no standard or complex mixture showed no mutagenicity. Changes in mutagenicity by acetylation The reaction rate of 2-aminoanthracene with acetic anhydride was monitored by TLC and the amide was identified by proton NMR. The reaction was greater than 95% complete in 6 h, as determined by the NMR spectra. The mutagenicities of the products of PAA and acetic anhydride were uniformly indirect. After acetylation, the mutagenicities of 5 of the 6 PAA standards were reduced to less than 21% (Table 3). However, the
than 2 times background.
activity of 1-acetylaminopyrene rose to more than 6 times the levels of the free amine. Acetylation of the coal oil sample with acetic anhydride reduced the observed mutagenicity to less than 30% of the unreacted value (Table 4). Again, reaction controls were similar to those in the sample in DMSO, and values were extracted from the linear portion of the dose-response curve. After acetylation, low Btu tar showed a decrease in mutagenicity of 52% (Table 3). By careful choice of sample workup, possible byproducts of the reaction, such as acetic anhydride and acetic acid, were removed. Reaction controls in which experiments were run without samples left no detectable mass after workup.
15
TABLE
4
EFFECT OF ADDED ON CELL VIABILITY Sample
BUFFER
pg/plate
AND
SODIUM
NITRITE
Viable count X lO*
DMSO
0.0
2.5 +0.2
Tar/DMSO
1 8
2.4+0.2 2.3kO.l
Tar/reaction
1 8
2.3 +0.3 2.0+0.1
Coal oil/DMSO
2 20
1.7+0.2 1.9AO.3
Coal oil/reaction
2 20
1.6+0.1 1.8+0.2
a Estimated from serial dilution of TA98. Determined with S9 metabolic enzymes. Sample controls are tar and coal oil in DMSO only. Mixtures after addition of buffer and sodium nitrite are entitled reaction.
Two reaction schemes were used to assess the contribution of PAA to the mutagenicity of tar trap tar from a low Btu gasification process. Nitrous acid was effective in eliminating nearly all of the S9-dependent mutagenic activity of PAA standards and a coal oil sample. Likewise, nitrous acid reduced the indirect mutagenicity of a low Btu coal gasifier tar, although 31% of the mutagenicity remained. Acetylation reduced the indirect mutagenicity to only 52% of the original. These results indicate that coal gasification tars, unlike coal liquefaction fuels, contain significant amounts of indirect-acting mutagens other than PAA. Nitrosation at low pH (0.3% HCl) generated substantial amounts of direct mutagenic activity in less than a minute reaction time. This particular acid concentration was originally chosen by Yoshida in his studies of protein pyrolysates because of its similarity to that of gastric solution (Yoshida and Matsumoto, 1978). Both Pelroy and Stewart (1982) and Haugen et al. (1981a), found this acid concentration useful in their respective studies of the reaction of nitrous acid with SRC-II liquids and BaP (Pelroy and Stewart 1982; Haugen et al., 1981a). In the present study, the mutagenic response of the gasifier tar at low pH was com-
plicated by the production of direct-acting mutagens. The choice of the proper pH range was based on studies of Tsuda et al. (1981) on the effect of nitrite on 2-amino-a-carbolene (2A-a-C). In that study, acid concentration was found to be important in determining the final products of the nitrosation reaction. Thus, at a pH of 1.1 the indirect activity of 2A-a-C was destroyed, but direct activity rose markedly. The compound 2-hydroxy-3-nitroso-a-carbolene, a direct mutagen, was isolated from the reaction product mixture. At a higher pH, 2A-WC is converted wholly to 2-hydroxy-a-carbolene, a nonmutagen. At the low pH value, the phenol formed probably activates the ring for attack by nitrosonium ion. The attack by nitrosonium is apparently less favored at a higher PH. Low Btu coal gasification tars are known to contain numerous phenols; therefore, formation of such nitroso compounds is not unlikely at low pH and could have been the source of the direct mutagenic activity observed. Acid concentrations of the reacting solution were run at two different pH values (2.5 and 4.0) and reaction times (0.5 and 5 h). At a pH of 2.5 and a reaction time of 0.5 h, the indirect rrtutagenicity of a PAA standard was destroyed, whereas no direct mutagenicity was generated in the tar. Cytotoxicity did not appear to be an explanation for loss of mutagenicity. The Salmonella TA98 cells survived treatment similar to those subjected to nitrous acid. Both Pelroy and Stewart (1982) and Yoshida and Matsumoto (1978) observed identical results. Likewise, treatment with nitrite alone will probably not cause cytotoxicity; previous studies (Pelroy and Stewart, 1982) have shown little induction of mutagenicity or cytotoxicity by nitrite. A second derivatization procedure was characterized to substantiate the results of nitrosation. This scheme was based on the reaction of acetic anhydride to primary amines. It was found that while most PAA are markedly reduced in mutagenic activity by acetylation, l-aminopyrene actually increases in mutagenicity by a factor of more than 6. This observation is of interest because 1-acetylaminopyrene has been observed as a metabolite of 1-nitropyrene in Salmonella bacteria
16
(Messier et al., 1985) and in perfused lungs (Bond, 1983). When acetylation was applied to a coal oil sample, most of the mutagenicity was destroyed. When low Btu tar was reacted with acetic anhydride, about one half of the mutagenic activity remained. While somewhat more mutagenicity is associated with low Btu tars treated with acetic anhydride than with nitrous acid, these results again indicate that, unlike coal oil, PS, and HD, other mutagens are important in determining the indirect mutagenicity of coal gasification tars. An estimated 30-50s of the mutagenicity is due to non-PAA chemicals when results of the two reactions are combined. Although the chemicals responsible for the nonPAA mutagenicity are unknown, one possibility is aza-arenes. Numerous aza-arenes have been positively identified in coal-related mixtures (Later et al., 1981), and many are known to be mutagenic (Dong et al., 1978). Mutagenic PAH have also been identified in coal-related mixtures, such as benzo[a]pyrene (Later et al., 1981). Further chemical and biological investigations into these compound classes are necessary to evaluate the contributions of these classes to the overall mutagenicity of coal tar.
mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Bond, J. (1983) Private communication. Dong, M., I. Schmeltz, E. Lavoic and D. Hoffman (1978) Aza-arenes in the respiratory environment: Analysis and assays for mutagenicity, in: P.W. Jones and R.I. Freulenthal (Eds.), Carcinogenesis: Polynuclear Aromatic Hydrocarbons, Vol. 3, Raven, New York, pp. 97-108. Haugen, D.A., M.J. Peak and C.A. Reilly Jr. (1981a) Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines, non-specific reactions with phenols and benzo[ a]pyrene, Mutation Res., 82, 59-67. Haugen, D.A., M.J. Peak, K.M. Suhrbler and V.C. Stamoudis (1981b) Isolation of mutagenic aromatic amines from a coal conversion oil by cation exchange chromatography, Anal. Chem., 54, 32-31. Later, D.W., M.L. Lee, K.D. Bartle, R.C. Kong and D.L. Vassilaros (1981) Chemical class separation and characterization of organic compounds in synthetic fuels, Anal. Chem., 53, 1612-1620. Later, D.W., M.L. Lee and B.W. Wilson (1982) Selective detection of amino polycyclic aromatic compounds in solvent refined coal, Anal. Chem., 54, 117-123. Messier, F., C. Lu, P. Andrews, B.E. McCarry, M.A. Quilliam and D.R. McCalla (1985) Metabolism of I-nitropyrene and for motion of DNA adducts in Salmonella typhimurium, in preparation. Newton, G.J. (1980) Low Btu Gasifier Emissions Toxicology Program Status Report, June 1980, Inhalation Toxicology Research Institute, LMF-77, Available from National Technical Information Service.
Acknowledgements
Pelroy, R.A., and D.L. Stewart (1982) The effects of nitrous acid on the mutagenicity of two coal liquids and their genetically active chemical fractions, Mutation Res.. 90, 297-308.
The authors thank Nicole Dumont for technical assistance. We also thank the personnel at the Morgantown Energy Technology Center and our colleagues at the Inhalation Toxicology Research Institute for helpful discussions. This research was performed under U.S. Department of Energy Contract DE-AC04-76EV 01013. References Ames, B.N., J. McCann
detecting
carcinogens
and E. Yamasaki (1975) Methods for and mutagens with the Salmonella/
Royer, R.E., C.E. Mitchell, R.L. Hanson, J.S. Dutcher and W.E. Bechtold (1983) Fractionation, chemical analysis and mutagenicity testing of low Btu coal gasifier tar, Environ. Res., in press. Tomkins, B.A., and C.-H. Ho (19820 Determination of polycyclic aromatic amines in natural and synthetic crudes, Anal. Chem., 54, 91-96 Tsuda, M., M. Nagao, T. Hirayama, and T. Sugimura (1981) Nitrite converts 2-amino-cY-carboline, a non-mutagen, and 2-hydroxy-3-nitroso-a-carboline, a direct mutagen, Mutation Res., 83, 61-68. Yoshida, D., and T. Matsumoto (1978) Changes in mutagenicity of protein pyrolyzates by reaction with nitrite, Mutation Res., 58, 35-40.
Research, 155 (1985) l-16
MTR 00939
Contribution
of primary
W.E. Bechtold,
aromatic amines to the mutagenicity and coal oils J.S. Dutcher,
of gasifier tars
A.L. Brooks and R.F. Henderson
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Albuquerque, NM 87185 (U.S.A.)
Research Institute, P.O. Box 5890,
(Received 10 December 1983) (Revision received 14 May 1984) (Accepted 14 September 1984)
Summary Two reactions that chemically alter primary aromatic amines (PAA) were used to assess the contribution of these compounds to the indirect bacterial mutagenicity of tar from an experimental low Btu gasifier. The first reaction, nitrosation, effectively eliminated the mutagenicity of several PAA standards and a coal oil when run in a low pH media (1.2). When applied to gasifier tar, extensive direct (not requiring metabolic activity) mutagenicity was generated. This direct mutagenicity limited the interpretation of results. When the pH of the reaction media was raised to 2.5, the mutagenicity of PA.4 standards and the coal oil were still greater than 90% eliminated, however, no direct mutagenicity was observed for the gasifier tar. Furthermore, only 61% of the indirect (requiring metabolic activation) mutagenicity was eliminated. Acetylation reduced the indirect activity of most primary amine standards by greater than 79%. Acetylation of the tar likewise eliminated part, but not all, of the activity, whereas most of the activity of the coal oil was eliminated. These results indicated that a much lower percentage of the mutagenic activity of low Btu coal tar samples was due to primary aromatic amines than was the case for coal oil.
Primary aromatic amines (PAA) are believed to contribute significantly to the observed bacterial mutagenicity and potential hazards associated with coal-derived synthetic fuels (Pelroy and Stewart, 1982). In principle, the relative contributions of PAA to the overall observed mutagenicity of complex mixtures could be determined by isolation of pure PAA fractions. Unfortunately, the careful and extensive separations necessary to isolate such pure PAA fractions have proved difficult and time-consuming because of their low concentrations in the complex mixtures and high chemical reactivities (Later et al., 1982; Ton&ins and Ho, 1982; Haugen et al., 1981b). Other means of assessing the contributions of PAA to the bacterial mutagenicity have therefore been sought. 0165-1218/85/$03.30.0
1985 Elsevier Science Publishers
Reaction with nitrous acid has been a successful technique for determining the contributions of PAA to the bacterial mutagenicity of process solvents (PS) and heavy distillates (HD) from solvent refined coal (SRC) (Pelroy and Stewart, 1982). Nitrous-acid-dependent decreases in indirect bacterial mutagenic activity (requiring rat-liver metabolic enzymes) were observed for numerous PAA standards, whereas mutagenic activity for non-PAA polycyclic aromatic hydrocarbons (PAH) and aza-arenes remained unchanged. The observation that more than 90% of the indirect activity for PS and HD disappears after reaction with nitrous acid led Pelroy and Stewart (1982) to conclude that PAA are quantitatively important in determining the mutagenicity of these mixtures. In
B.V. (Biomedical
Division)
8
their study, increases in direct mutagenicity accompanied decreases in indirect mutagenicity. Although slight in the case of PS and HD, side reactions creating direct-acting mutagens such as nitroso and nitro PAH may complicate the interpretation of the effect of nitric acid on other complex mixtures. Thus, Haugen et al. (1981a) developed methods for nitrous acid treatment specifically designed to minimize these side reactions. Under their conditions, generation of direct activity by reaction of nitrous acid with benzo[alpyrene (BaP) was shown to be minimal, whereas the indirect mutagenic activity of 2aminoanthracene was decreased to 5% of its original value. The contribution of PAA to the mutagenic activity of low Btu gasifier tars is also of interest because the basic fraction in which PAA would be found has high specific indirect mutagenic activity (Royer et al., 1983). We report two methods to assess the contribution of PAA to the mutagenicity of tar from an experimental low Btu gasifier. For validation of the methods, a coal oil similar in nature to SRC-II liquids was used for comparison. The first method, nitrous acid treatment, was shown to generate extensive direct activity in the gasifier tar using the conditions of Haugen et al. (1981a). At a higher pH, generation of direct activity was minimized, whereas indirect activity was significantly but not totally decreased. The second method, acetylation, also did not eliminate the indirect activity completely. These two methods indicated that PAAs account for only part of the observed mutagenic activity in low Btu coal tar.
(CRM) acquired from the Fossil Fuels Research Matrix Program (Oak Ridge National Laboratory). Methylene chloride, benzene, and dimethyl sulfoxide (DMSO) were purchased from Burdick and Jackson Laboratories. Pyrene, 2-aminoanthracene, 2-aminofluorene, 3-aminofluoranthene, laminopyrene, 6-aminochrysene, 9-aminophenanthrene, carbazole, and phenanthridine were obtained from Aldrich, 2-Aminofluorene, 6-aminochrysene, and l-aminopyrene in DMSO showed some direct bacterial mutagenic activity, and gas chromatography/mass spectroscopy (GC/ MS) showed contamination from the corresponding nitro derivatives. These three amines were therefore purified by preparative silica gel thin-layer chromatography (TLC). TLC plates were eluted with a 3 : 1 mixture of benzene: methylene chloride, scraped, and the silica gel extracted by sonication with methylene chloride. Purified compounds showed no traces of nitro derivatives by GC/MS analysis, and no direct mutagenic activity was detected. Mutagenesis assay Chemical standards, samples, and reaction products were tested for biological activity in Salmonella typhimurium strain TA98 as previously published (Ames et al., 1975). Each concentration was tested in triplicate with 5 concentrations per chemical. The mutagenic activities of the chemicals were evaluated both with (indirect) and without (direct) Aroclor-125Cinduced rat liver homogenate fraction (S9) as a source of metabolic enzymes. The number of revertant colonies was
\ /b
Materials and methods Materials Gasifier tars were collected from the tar trap of an experimental low Btu gasifier unit at the Morgantown Energy Technology Center, Morgantown, WV (METC). The coal gasifier was a stirred-bed unit using a modified Lurgi process. The gasifier and cleanup system have been described elsewhere (Newton, 1980). The tar trap was positioned in the process stream following a cyclone, which removed ash, and a humidifier which cooled and condensed the tars. Coal oil samples were comparative research materials
H,O
.N2@
Acetic NH2
Pg. 1. Reactions anhydride.
Anhydride of PAA with (A) nitrous
Y-KCH3 H 0 acid and (B) acetic
9
counted on an Artek Model 800 automatic colony counter and related to the concentration in the bacterial plate using a linear least squares regression analysis. The biological activity is thus reported as revertants/pg extract using the linear portion of the dose-response curve. The linear portion of the curve was defined in every case by a minimum of 3 points. Acceptability of points was based on arriving at a correlation coefficient of 0.95 in the regression analysis. Values used in direct comparisons were derived on the same days to minimize day-to-day variation in the tester strain. Nitrosation reactions Treatment of primary aromatic amines with nitrite in acid results in the formation of a diazonium salt, followed by decomposition to a phenol (Fig. 1). Initial nitrosation reaction conditions (method I) were essentially those of Haugen et al. (1981a), with only slight variations. About 5-10 mg of sample was dissolved in 0.5 ml of DMSO and added to a solution of NaNO, (5 mg/ml) in HCl (0.3%, pH 1.2). For the control, NaNO, was omitted. Reactions were allowed to proceed for 2 min at room temperature and were stopped by the addition of 1.5 ml of 1 N NaHCO,. The materials were lyophilized overnight and then dissolved (suspended) in DMSO for bacterials mutagenicity testing. Nitrosation reactions were also produced by methods similar to those of Tsuda et al. (1981) (method II). Typically, PAA or complex mixtures were dissolved in DMSO to concentrations of 1 mg/ml and 10 mg/ml respectively. Aliquots of 0.4 ml each of the DMSO mixtures were added to 3.2 ml of citrate/Na,HPO, buffer. Citrate (50 mM) and phosphate (50 mM) buffers were mixed to form solutions of pH 2.5 and 4.0. Nitrosation reactions were conducted by adding 0.4 ml sodium nitrite solution (10 mM). As controls, either 0.4 ml of buffer was added, or nitrite was added without mutagens. Reactions were terminated by the addition of 0.2 ml of 0.25 M sodium bicarbonate to 0.8 ml of the reaction mixtures after 0.5-5 h. All solutions were filter sterilized before use in the Ames test. Acetylation reactions Treatment of PAA with acetic anhydride
results
in the acetylation of primary amines to amides (Fig. 1). For acetylation, 10 mg of standard or complex mixture was dissolved in 4 ml of a 20 : 1 solution of methylene chloride in pyridine, and 50 ~1 of acetic anhydride was added. Again, as a control, acetic anhydride was omitted. Reactions were stopped after 6 h by the addition of 0.6 ml of 1 N NH,OH, followed by enough ethanol to produce a homogenous solution. The solvents were evaporated under a stream of nitrogen, and the ammonium acetate was removed by sublimation in vacua at room temperature. Results
Changes in mutagenicity by nitrosation (method I) As observed by Haugen et al. (1981a), nitrosation by method I was effective in reducing the indirect bacterial mutagenicity of several PAA standards to less than 10% of the unreacted value. Furthermore, nitrosation under these conditions reduced the indirect bacterial mutagenicity of the coal oil sample to less than 10% within 3 min of the onset of the reaction without the generation of direct mutagenicity. When applied to tar from the low Btu gasifier, no reduction in the indirect muta-
15
. i
l =with
S-9
A= without
S-9
I
!
I
I
I
2
4
6
8
10
TIME (mid Fig. 2. Kinetics of reaction of gasifier tar trap tar with nitrous acid (method I). Conditions: n, with S9; A, without S9.
10
genie activity was noted (Fig. 2). This may be due to the simultaneous generation of direct mutagenic activity. In fact, within 20 set of the onset of the
reaction almost 2 revertants/pg of direct activity were generated, and direct activity continued to rise with time, as did the indirect activity.
TABLE 1 TA98 MUTAGENICITY OF 2-AMINOANTHRACENE (2AA) AND GASIFIER TAR TRAP TAR (REVERTANTS/ng) FUNCTION OF pH AND TIME OF NITROUS ACID TREATMENT (METHOD II) Time (h)
0.0
a
Sample
2AA
Amount/
pHb
plate (ng)
2.5
0.0 0.1 0.2 0.4 0.8 rev/ng
Tar 2.0 4.0 8.0 16.0 rev/ng 0.5
2AA
0.0 0.1 0.2 0.4 0.8 rev/P g
Tar
0.0 2.0 4.0 8.0 16.0 rev/P g
5.0
2AA
0.0 0.1 0.2 0.4 0.8 rev/pg
Tar
0.0 2.0 4.0 8.0 16.0 rev/pg
4.0
-s9
+s9
16 +3 26 *3 22 *2 18.2*2 21 56 l>
25 99 233 542 988 1271
0.0 17 21 22 20 l>
AS A
*2 +4 *3 *5
16 25 25 17 22 1>
*3 *1 *l *3 +3
16 18 29 31 37 l>
-13 f0 *1 *2 *7
16 18 18 21 22 l>
&3 rtl *O *o *3
16 *3 30 *2 48 *4 70 zt8 87 *l 3.8rtO.9
-s9 + 2 f 4 f 1 *51 k92’ *69
16 k3 110 *lo 179 *22 244 *20 339 *29 15.4+ 2.3 25 27 39 35 66 51
f 2 f 3 f 1 + 6 f lC *14
25 f 2 55 f 8 77 *18 96 +ll 133 +18” 5.3+ 0.6 25 23 38 44 55 40
+ 2 + 5 k10 +3 *14= *11
25 + 2 56 *16 78 f 9 110 *13 149 529” 6.4* 0.8
16+ 26+ 16& 17+ 16f 1>
+s9 3 7 3 1 6
25 f 2 22+ 3 13* 4 22k 8 35* 5 l>
25 97 267 977 1308 1763
16+ 59 f 109 f 186 f 349 f 20.5+
16+ 24k ll& 24+ 19* 12
3 1 2 1 1
25 98 269 773 1321 1762
16+ 14* 15+ 22+ 21+ 1>
3 3 2 3 8
25 91 170 216 292 13.0
16+ 3 19*11 22* 2 22+ 5 22*17 1>
25 69 131 393 932 1266
16& 22& 18+ 18* 29+ 12
3 2 9 1 2
f 2 f 5 f 11 + 6 + 61’ k413 325 + 11 1 13 53 0.4
f 2 f 1 f 28 f 77 k117’ 5173 f 2 + 11 f 16 k 8 f 18’ + 2.8 + k f f + f
2 17 54 30 57 68
25 f 2 58 f 6 80 + 6 196 +124 229 f 6 12.6+_ 3.7
’ Sample control - determined with buffer at pH 2.5 and 4.0, but without nitrite. b Determined without (- S9) and with (+ S9) Aroclor-1254-induced rat-liver microsomes (9000 X g fraction). ’ Significance of mean response established by detection of mutagenicity at greater than 2 times background.
2
11 TABLE 2 MUTAGENICITY OF STANDARDS NITROUS ACID = Chemical
Amount/ plate ( c1g)
2-Aminoanthracene
0
0.1 0.2 0.4 0.8 rev/pg 3-Aminofluoranthene
0 0.1 0.2 0.4 0.8 1.0 rev/pg
2-Aminofluorene
0 0.1 0.2 0.4 0.8 1.0 rev/p g
6-Aminochrysene
0 0.1 0.2 0.4 0.5 rev/yg
9-Aminophenanthrene
0 0.1 0.2 0.4 0.8 1.0 rev/p g
l-Aminopyrene
0 0.05 0.1 0.2 0.4 0.5 rev/y g
Carbazole
0 50 100 200
AND COMPLEX
MIXTURES
Control b (rev/pg) -s9
Reaction (rev/pg)
+s9
16+ 26+ 22* 18k 21+l>
3 3 2 2 6
17+ 28k 20* 31* 36+ 65k l>
2 3 7 4 3 6
19+ 23k 26k 18* 21* 29+ l>
3 7 2 5 7 1
24*11 21* 3 17* 4 16k 2 22* 9 12 19+ 16f 20+ 20f 26+ 27k l>
3 2 4 2 5 5
27k 20+ 24+ 24k 18k 23* l>
5 9 6 3 2 5
24k 30+ 24+ 32k 12
1 8 5 6
IN TA98 BEFORE AND AFTER TREATMENT
-s9
+s9
25 99 233 542 988 1271
+ 2 f 4 f 1 f 51 f 92’ f 69
16 25 25 17 22 l>
f f f + f
24 49 73 169 555 872 904
f 4 f 6 f 4 f 22 f 41 f 71’ *110
17 20 20 21 22 35 l>
+ f + + f f
32 + 3 49 f 4 62 f 7 91 f 9 153 f 8 208 f 34’ 171 f 12
19 23 19 26 30 30 l>
+3 f f f + *
f 1 f 8 f 12 k263 k166” f 774
24 27 19 21 20 l>
f + * f f
f f f f f f f
3 8 5 21 32 47’ 54
19 19 24 23 18 30 l>
+ f f + f f
39 f 2 38 + 9 52 f 14 66 f 2 120 f 10 150 f 13c 245 f 13
27 17 19 22 18 24 l>
f f f + + +
78 37 84 1474 1889 5094 32 38 55 114 421 518 570
28 33 35 33 l>
f f f f
1 4 16 6
24 25 54 113 l>
Remaining (%) d
3 1 1 3 3
2 5 1 4 4 7
4 3 2 1 3
1 4 2 3 3
3 6 7 5 1 3
5 1 8 5 2 5
f 11 +_ 4 f 13 + 7
25 27 39 35 66 51
f 2 f 3 + 1 zt 6 f lC * 14
24 23 26 35 33 31 l>
f 4 f 8 + 7 f 10 f 4 + 1
32 35 29 35 39 39 l>
+ f f f * f
28 35 3i 176 295 659
4
0 3 8 8 8 3 2 0 f 1 + 3 f 10 f 14 + 31’ k131
32 27 30 31 35 32 l>
f f + + * +
39 32 32 31 28 31 l>
f 2 f 1 f 9 + 6 + 10 f 6
28 37 51 79 l>
f f f f
13
3 1 2 7 9 5 0
0 1 3 6 5 0
WITH
12
TABLE
2 (continued)
Chemical
Amount/ plate (as)
Phenanthridine
Coal oil A
Tar trap tar
’ b ’ d
Control -s9
b (rev/p&
Reaction
+s9
0 2 4 8 16 20 rev/u g
24*11 21* 14* 17* 17+ 14+ l>
0 2 4 8 16 20 rev/ng
21* 25? 25+ 19* 21* 28+ I>
2 7 4 1 4 1
0 1 2 4 8 10 rev/pg
21* 16+ 24k 18* 22+ 19* 11
2 1 5 1 5 1
I 5 2 1 4
(rev/p@
-s9
28 + 1 24 rt -I 34 * I 54 * 9 155 * 11’ 152 + 21 9.5* 1.4
24 13 15 18 16 11 l>
32 + 47 k 83 k 149 * 324 + 198 ; 19.8k
4 6 14 15 40” 17 0.9
32 + 4 39 f 3 68 * 8 123 f 15 156 + 11 185 f 12’ 15.25 2.1
Remaining
(%) d
+s9 kll f f f k f
28 * 31 f 50 * 61 f 162 f 199 * 9.5*
1 1 17 26 23r 14 0.7
100
21 f 2 31 f 5 26 f 2 31 f 7 53 *lo 87 f 9’ 3.6* 0.9
32 f 42 f 35 f 40 * 72 f 61 + 1.7*
4 4 2 7 19 9’ 0.6
9
21 24 24 24 35 35 l>
32 * 38 + 32 f 63 f 78 ? 83 k 5.9?
4 1 4 6 6 3’ 1.6
39
_t + + + k *
5 I 7 3 9
2 4 3 8 6 8
Without (- S9) and with ( + S9) Aroclor-1254-induced rat-liver microsomes (9000 X g fraction). Control reactions have buffer but no sodium nitrite added. Significance of mean response established by detection of mutagenicity at greater than 2 times background. Ratio of mutagenicities before and after reaction, with S9.
Changes in mutagenicity by nitrosation (method II) Nitrosation reaction conditions were sought that would reduce the indirect mutagenic activity of PAA standards without generation of direct activity for tar trap tar. Time (0.5 and 5 h) and pH (2.5 and 4) were the two variables investigated. Results in Table 1 show that, regardless of the time of reaction, at pH 4, nitrous acid had limited effect in reducing the indirect activity of 2-aminoanthracene. At pH 2.5, 5 h of reaction time lowered the mutagenicity of 2-aminoanthracene to 2% of the unreacted standard. However, 3.8 revertants/pg of direct activity were also generated. The reaction time of 0.5 h at pH 2.5 proved optimum for reducing the indirect mutagenic activity of the aromatic amine standards without inducing direct mutagens. The indirect activity for the 2-aminoanthracene was reduced by 95%, but not direct activity was generated. Reaction conditions of pH 2.5 and 0.5 h were used to investigate the effect of
nitrous acid on several standards (Table 2). In all cases, nitrous acid reduced the mutagenicities of PAA standards to less than 16% of the control value. Mutagenicities for a secondary amine, carbazole, and an aza-arene, phenthridine, were not affected by nitrous acid treatment. When the coal oil sample was subjected to HCl without NaNO,, little change in the mutagenicity was noted (Table 2). However, addition of nitrite reduced the indirect activity to 9%, the value observed in HCl alone. Only a small increase in direct-acting mutagenicity was detected after treatment with nitrous acid. Treatment of tar trap tar with nitrous acid decreased but did not eliminate the indirect mutagenicity (Table 2). Thus, after treatment, 39% of the mutagenicity remained. The administration of HCl/NaNO1 had no effect on cell viability either in conjunction with PAA standards or with complex mixtures (Table 4). Viability studies were run
13 TABLE
3
MUTAGENICITY OF STANDARDS ACETIC ANHYDRIDE = Chemical
AND
Amount/
COMPLEX
Control
MIXTURES
b
IN TA98 BEFORE
Reaction
(rev/p@
AND
AFTER
Remaining
plate (PP) 2-Aminoanthracene
0.0 0.1 0.2 0.4 0.8 1.0 rev/y g
3-Aminofluoranthene
0.0 0.05 0.1 0.2 0.4 0.8 rev/pg
2-Aminofluorene
0.0 0.05 0.1 0.2 0.4 0.8 rev/pLg
6-Amincchrysene
0.0 0.1 0.2 0.4 0.8 1 .o rev/B g
9-Aminophenanthrene
0.0 0.025 0.05 0.1 0.2 0.4 rev/p g
I-Aminopyrene
0.0 0.025 0.05 0.10 0.20 0.40 rev/p g
1-Aminopyrene (repeat)
0.0 0.05 0.1 0.2 0.4 rev/p g
38 50 95 303 846 1246 1330
f 3 f 12 f 19 + 17 * 30 +261c +105
38 30 32 47 71 115 88
+ + f f * f f
3 6 7 18 19 30= 15
30 27 34 88 252 821 114
+ 8 k 3 * 10 * 1 f 17 +105’ k129
30 41 48 65 131 203 224
+ f k f + f k
8 10 4 14 12 19’ 15
20
30 27 47 72 110 246 284
f 8 + 12 * 4 + 5 f 9 + 8’ k 18
+ +
8 9 9 1 8 2’ 8
21
30 42 66 268 1570 175 2264
* 8 f 2 * 13 + 51 f 77c f 44 k462
30 41 38 42 75 164 374
f 8 f 6 + 3 f 9 * 12 + 22’ * 40
30 29 37 46f 59 105 196
+ f +
30 30 45 63 86 153
30 28 40* 42 47 78 59 30 25 31 33 43 179 133
+ f * f
TREATMENT
k 8 +_ 7 * 4 f 5 f 2 + 36’ + 61
30 30 30 27 21 30 12
k 8 + 14 f 10 k 2 f 3 5 6
8 2 5 6 + 5 + 6’ f 12
30 106 200 340 557 592 1254
* 8 * 15 * 10 f 19 + 24 +165’ *364
640
+ 8 f 3 + 4 f 10 f 7c f 21
30 179 320 488 635 1230
+ 8 f 13 f 30 + 40 f 4’ +254
804
(I%)
WITH
14
TABLE
3 (continued)
Chemical
Amount/
Control
h
31 23 24 31 12
* + k k
Reaction
(rev/p@
Remaining(W)
plate (p 9) Carbazole
0.0 25 50 100
rev/pg Phenanthridine
0.0 L
4 8 16 20 rev/p g Coal oil A
0 2 4 8 16 20 rev/p g
Tar trap tar
0.0 1
i 4 8 10 rev/p g
36 f 28 f 44 * 70 f 109 f 138 * 5.9*
5 2 3 6
31 26 30 38 1>
* * * f
5 1 6 2
1 9 4 3 10 20’ 0.2
36 f 1 32 f 6 42 + 5 59 f 8 110 f 19 120 * 19’ 5.1+ 0.3
86
28 f 1 30 f 2 61 * 8 79 * 15 189 + 7 212 * 22’ 10.4+ 0.8
28 f 1 27 f 10 30 k 6 46 + 13 70 * 3 82 * 7’ 3.1+ 0.1
30
31 * 42 k 61 * 120 f 131 * 162 + 12.2*
31 + 7 38 f 2 42 + 2 62 f 11 89 f 10 89 k 11’ 6.3* 0.8
52
7 6 5 4 5 17’ 2.8
a With Aroclor-1254-induced rat-liver microsomes (9000 X g fraction). b Control reaction, with methylene chloride and pyridine, without acetic anhydride. ’ Significance of mean response established by detection of mutagenicity at greater
simultaneously with mutagenicity and used the same mixtures at the same concentrations. Furthermore, controls using nitrite but no standard or complex mixture showed no mutagenicity. Changes in mutagenicity by acetylation The reaction rate of 2-aminoanthracene with acetic anhydride was monitored by TLC and the amide was identified by proton NMR. The reaction was greater than 95% complete in 6 h, as determined by the NMR spectra. The mutagenicities of the products of PAA and acetic anhydride were uniformly indirect. After acetylation, the mutagenicities of 5 of the 6 PAA standards were reduced to less than 21% (Table 3). However, the
than 2 times background.
activity of 1-acetylaminopyrene rose to more than 6 times the levels of the free amine. Acetylation of the coal oil sample with acetic anhydride reduced the observed mutagenicity to less than 30% of the unreacted value (Table 4). Again, reaction controls were similar to those in the sample in DMSO, and values were extracted from the linear portion of the dose-response curve. After acetylation, low Btu tar showed a decrease in mutagenicity of 52% (Table 3). By careful choice of sample workup, possible byproducts of the reaction, such as acetic anhydride and acetic acid, were removed. Reaction controls in which experiments were run without samples left no detectable mass after workup.
15
TABLE
4
EFFECT OF ADDED ON CELL VIABILITY Sample
BUFFER
pg/plate
AND
SODIUM
NITRITE
Viable count X lO*
DMSO
0.0
2.5 +0.2
Tar/DMSO
1 8
2.4+0.2 2.3kO.l
Tar/reaction
1 8
2.3 +0.3 2.0+0.1
Coal oil/DMSO
2 20
1.7+0.2 1.9AO.3
Coal oil/reaction
2 20
1.6+0.1 1.8+0.2
a Estimated from serial dilution of TA98. Determined with S9 metabolic enzymes. Sample controls are tar and coal oil in DMSO only. Mixtures after addition of buffer and sodium nitrite are entitled reaction.
Two reaction schemes were used to assess the contribution of PAA to the mutagenicity of tar trap tar from a low Btu gasification process. Nitrous acid was effective in eliminating nearly all of the S9-dependent mutagenic activity of PAA standards and a coal oil sample. Likewise, nitrous acid reduced the indirect mutagenicity of a low Btu coal gasifier tar, although 31% of the mutagenicity remained. Acetylation reduced the indirect mutagenicity to only 52% of the original. These results indicate that coal gasification tars, unlike coal liquefaction fuels, contain significant amounts of indirect-acting mutagens other than PAA. Nitrosation at low pH (0.3% HCl) generated substantial amounts of direct mutagenic activity in less than a minute reaction time. This particular acid concentration was originally chosen by Yoshida in his studies of protein pyrolysates because of its similarity to that of gastric solution (Yoshida and Matsumoto, 1978). Both Pelroy and Stewart (1982) and Haugen et al. (1981a), found this acid concentration useful in their respective studies of the reaction of nitrous acid with SRC-II liquids and BaP (Pelroy and Stewart 1982; Haugen et al., 1981a). In the present study, the mutagenic response of the gasifier tar at low pH was com-
plicated by the production of direct-acting mutagens. The choice of the proper pH range was based on studies of Tsuda et al. (1981) on the effect of nitrite on 2-amino-a-carbolene (2A-a-C). In that study, acid concentration was found to be important in determining the final products of the nitrosation reaction. Thus, at a pH of 1.1 the indirect activity of 2A-a-C was destroyed, but direct activity rose markedly. The compound 2-hydroxy-3-nitroso-a-carbolene, a direct mutagen, was isolated from the reaction product mixture. At a higher pH, 2A-WC is converted wholly to 2-hydroxy-a-carbolene, a nonmutagen. At the low pH value, the phenol formed probably activates the ring for attack by nitrosonium ion. The attack by nitrosonium is apparently less favored at a higher PH. Low Btu coal gasification tars are known to contain numerous phenols; therefore, formation of such nitroso compounds is not unlikely at low pH and could have been the source of the direct mutagenic activity observed. Acid concentrations of the reacting solution were run at two different pH values (2.5 and 4.0) and reaction times (0.5 and 5 h). At a pH of 2.5 and a reaction time of 0.5 h, the indirect rrtutagenicity of a PAA standard was destroyed, whereas no direct mutagenicity was generated in the tar. Cytotoxicity did not appear to be an explanation for loss of mutagenicity. The Salmonella TA98 cells survived treatment similar to those subjected to nitrous acid. Both Pelroy and Stewart (1982) and Yoshida and Matsumoto (1978) observed identical results. Likewise, treatment with nitrite alone will probably not cause cytotoxicity; previous studies (Pelroy and Stewart, 1982) have shown little induction of mutagenicity or cytotoxicity by nitrite. A second derivatization procedure was characterized to substantiate the results of nitrosation. This scheme was based on the reaction of acetic anhydride to primary amines. It was found that while most PAA are markedly reduced in mutagenic activity by acetylation, l-aminopyrene actually increases in mutagenicity by a factor of more than 6. This observation is of interest because 1-acetylaminopyrene has been observed as a metabolite of 1-nitropyrene in Salmonella bacteria
16
(Messier et al., 1985) and in perfused lungs (Bond, 1983). When acetylation was applied to a coal oil sample, most of the mutagenicity was destroyed. When low Btu tar was reacted with acetic anhydride, about one half of the mutagenic activity remained. While somewhat more mutagenicity is associated with low Btu tars treated with acetic anhydride than with nitrous acid, these results again indicate that, unlike coal oil, PS, and HD, other mutagens are important in determining the indirect mutagenicity of coal gasification tars. An estimated 30-50s of the mutagenicity is due to non-PAA chemicals when results of the two reactions are combined. Although the chemicals responsible for the nonPAA mutagenicity are unknown, one possibility is aza-arenes. Numerous aza-arenes have been positively identified in coal-related mixtures (Later et al., 1981), and many are known to be mutagenic (Dong et al., 1978). Mutagenic PAH have also been identified in coal-related mixtures, such as benzo[a]pyrene (Later et al., 1981). Further chemical and biological investigations into these compound classes are necessary to evaluate the contributions of these classes to the overall mutagenicity of coal tar.
mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Bond, J. (1983) Private communication. Dong, M., I. Schmeltz, E. Lavoic and D. Hoffman (1978) Aza-arenes in the respiratory environment: Analysis and assays for mutagenicity, in: P.W. Jones and R.I. Freulenthal (Eds.), Carcinogenesis: Polynuclear Aromatic Hydrocarbons, Vol. 3, Raven, New York, pp. 97-108. Haugen, D.A., M.J. Peak and C.A. Reilly Jr. (1981a) Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines, non-specific reactions with phenols and benzo[ a]pyrene, Mutation Res., 82, 59-67. Haugen, D.A., M.J. Peak, K.M. Suhrbler and V.C. Stamoudis (1981b) Isolation of mutagenic aromatic amines from a coal conversion oil by cation exchange chromatography, Anal. Chem., 54, 32-31. Later, D.W., M.L. Lee, K.D. Bartle, R.C. Kong and D.L. Vassilaros (1981) Chemical class separation and characterization of organic compounds in synthetic fuels, Anal. Chem., 53, 1612-1620. Later, D.W., M.L. Lee and B.W. Wilson (1982) Selective detection of amino polycyclic aromatic compounds in solvent refined coal, Anal. Chem., 54, 117-123. Messier, F., C. Lu, P. Andrews, B.E. McCarry, M.A. Quilliam and D.R. McCalla (1985) Metabolism of I-nitropyrene and for motion of DNA adducts in Salmonella typhimurium, in preparation. Newton, G.J. (1980) Low Btu Gasifier Emissions Toxicology Program Status Report, June 1980, Inhalation Toxicology Research Institute, LMF-77, Available from National Technical Information Service.
Acknowledgements
Pelroy, R.A., and D.L. Stewart (1982) The effects of nitrous acid on the mutagenicity of two coal liquids and their genetically active chemical fractions, Mutation Res.. 90, 297-308.
The authors thank Nicole Dumont for technical assistance. We also thank the personnel at the Morgantown Energy Technology Center and our colleagues at the Inhalation Toxicology Research Institute for helpful discussions. This research was performed under U.S. Department of Energy Contract DE-AC04-76EV 01013. References Ames, B.N., J. McCann
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Royer, R.E., C.E. Mitchell, R.L. Hanson, J.S. Dutcher and W.E. Bechtold (1983) Fractionation, chemical analysis and mutagenicity testing of low Btu coal gasifier tar, Environ. Res., in press. Tomkins, B.A., and C.-H. Ho (19820 Determination of polycyclic aromatic amines in natural and synthetic crudes, Anal. Chem., 54, 91-96 Tsuda, M., M. Nagao, T. Hirayama, and T. Sugimura (1981) Nitrite converts 2-amino-cY-carboline, a non-mutagen, and 2-hydroxy-3-nitroso-a-carboline, a direct mutagen, Mutation Res., 83, 61-68. Yoshida, D., and T. Matsumoto (1978) Changes in mutagenicity of protein pyrolyzates by reaction with nitrite, Mutation Res., 58, 35-40.