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Genetic activity in Saccharomyces cerevisiae of compounds found in effluents of pulp and paper mills
Mutation Research, 155 (1985) 53-60 Elsevier
53
MTR 00952
Genetic activity in Saccharomyces cereuisiae of compounds effluents of pulp and paper mills Earle R. Nestmann a Mutagenesis
found in
a* and Ernie G.-H. Lee b
Section, Environmental and Occupational Toxicology Division, Deparlment of National Health and Welfare, Ottawa, Ontario KlA OL2 and b B.C. Research, 3650 Wesbrook Mall, Vancouver, BC, V6S 2L2 (Canada) (Received (Accepted
11 September 14 September
1984) 1984)
Summary 20 compounds
in pulp mill effluents were screened for genetic activity in growing cells using D7 and XV185-14C without and with S9. Nine compounds were positive in one or the other yeast strain (7 in D7; 2 in XV18514C). One additional compound showed weak effects and two others showed elevated frequencies/survivor without absolute increases of mutants. The presence of S9 enabled detection of one positive and two weak effects, it enhanced the genetic activity of one compound in each strain, and it reduced the mutagenic effects of 4 others in strain D7. 7 of the 20 chemicals tested have been shown previously to be mutagenic in the Salmonella/mammalian-microsome assay. Of the 7 bacterial mutagens, 6 were positive and 1 had a weak effect in yeast. Saccharomyces
identified
cerevisiae strains
Recent international collaborative studies have shown that yeast assays are not only promising predictors of carcinogenic activity (de Serres and Hoffmann, 1981), but that they compare favorably in performance with other eukaryotic test systems in correctly identifying selected chemical carcinogens (Parry, 1985). The literature contains an increasing number of examples of mutagens that were detected in yeast but not in prokaryotes (Hannan and Nasim, 1978; Simmon, 1979; Nestmann et al., 1981; Nestmann and Lee, 1983), leading many laboratories of genetic toxicology to adopt Saccharomyces cerevisiae as a useful test. The present study continues our efforts to characterize the genetic toxicity associated with ef-
* Correspondence and reprint requests should be addressed to Dr. Earle R. Nestmann, Environmental Health Centre, Tunney’s Pasture, Ottawa KlA 0L2 (Canada). 0165-1218/85/$03.30
0 1985 Elsevier Science Publishers
fluents from pulp and paper mills. Such environmental mixtures are studied with different approaches; for example, by testing the complex mixture (or extracts from it) or by assessing its constituent compounds individually (Nestmann and Douglas, 1981). Previous reports have shown that effluent from pulp and paper mills induces genetic effects in a variety of test organisms (Ander et al., 1977; Eriksson et al., 1979; Douglas et al., 1980, 1983, 1984), including yeast (Kamra et al., 1983). Another strategy to identify compounds that contribute to the mutagenicity of the mixture is its sequential sub-fractionation using mutagenic activity as a means of following the active fractions (i.e., mutagenicity-directed fractionation; Douglas et al., 1984). This approach may be misleading, due to many possible interactions among the mutagens and non-mutagens present, or difficult, if mixtures contain a large number of mutagens. Many compounds in pulp and paper
B.V. (Biomedical
Division)
54
mill effluent have been shown to be mutagenic in Salmonella (Bjorseth et al., 1979; Nestmann et al., 1979, 1980; Rapson et al., 1980; Douglas et al., 1983), in yeast (Nestmann and Lee, 1983), and in mammalian cells (Ellenton et al., 1981; Douglas et al., 1983). In the present study, 20 compounds were assessed for reverse mutation and gene conversion in growing cultures of yeast. A preliminary report of this study was presented at the 11th International Conference on Yeast Genetics and Molecular Biology in Montpellier, France (September 1982). Yeast strains D7 (Zimmermann et al., 1975) and XV18514C (von Borstel et al., 1981) were used for testing according to the method described by von Borstel et al. (1981) using exponential phase cultures for Trp+ gene convertants or for reversion of the histidine, homoserine and tryptophan markers, respectively. In XV18514C, the trpS-48 mutation is ochre-suppressible and, therefore, can be reverted by mutations either within the gene or in a suppressor; the his1 -7 marker is a missense mutation and is relatively sensitive since it reverts largely at secondary sites; and there is evidence that the hom3-IO mutation is reverted by frameshift mutations (Mehta and von Borstel, 1981). Cells from frozen YEPD cultures (- SO’C) that were selected after preliminary screening for low mutation frequencies, were streaked on YEPD agar plates. After 2 days (3O”Q liquid subcultures (50 ml) were grown for 16 h in a gyratory water bath (30°C). For the tests, these growing cells were centrifuged and resuspended in YEPD (1 X 10’ cells/ml) with 25% of either phosphate buffer or S9 mix. Samples (up to 25 ~1) of solvent or test compound were combined with 2 ml of the cell suspension (+ S9) in a screw-top tube (16 X 125 mm) and incubated for 16 h either in a 30°C gyratory water bath ( - S9) or in a 37’C reciprocal water bath (+ S9). Treated cultures were chilled, washed, diluted 2-fold in phosphate buffer, and plated (0.2 ml/plate) on omission media for convertants and revertants. Further dilutions (10’) were made for survival determinations. All platings were made in triplicate, and the plates were incubated for 4 days (2 days for survival). Each experiment included 2 negative and 2 positive controls: nothing added to the culture and solvent;
ethyl methanesulfonate and 2-aminoanthracene (for S9). A response was considered to be positive when the numbers of mutants were higher than on the control plates and when the calculated mutant frequency (per lo5 survivors) was at least double the solvent control (see legend to Table 1). Dichloroacetone, succinic acid and methyl succinic acid were dissolved in water. The other 17 compounds were either diluted or dissolved in dimethyl sulfoxide (DMSO). Table 1 is a summary of the positive, weakly positive, questionable, and negative responses found. 7 compounds were found to induce Trp+ gene convertants in strain D7. None of these 7 was considered positive at any of 3 loci in haploid strain XV185-14C, although weak effects were found. 2 other chemicals induced His’ or Trp+ revertants in strain XV185-14C. As complementary yeast tests, these yeast strains detected a total of 9 positive compounds. 1 compound (bromodichloromethane) induced weak effects in both tests. 2 others (3- and 4-chloro-benzyl alcohol), showed higher frequencies of convertants or revertants per survivor without increases in numbers of mutant colonies per plates. Data for these positive, weak and questionable responses are given in Table 2. Of the 9 compounds found to have genetic activity, 6 have been been reported previously to be positive in the Salmonella assay, 1 was negative (in strain TA1535 only), and 2 are untested. 1 compound that had weak effects in yeast was positive in bacteria only when tested in a desiccator as an exposure chamber. Negative results for both Saccharomyces and Salmonella were observed for 5 compounds. Details of these comparisons and references are shown in Table 3. In comparison with a previous study of the genetic activity of 42 compounds in the same yeast strains (Nestmann and Lee, 1983) a much larger proportion of mutagens (9 of 20, or 45%) were found in this study than before (8 of 42, or 19%). However, the choices of compounds for each study were not random. Previous studies of these groups of compounds in Salmonella showed different proportions of bacterial mutagens, 35 and 7% respectively. The experimental protocols differed slightly in the two yeast studies (Table 4), and the question of what protocol is optimal in screening for
55 TABLE
1
SUMMARY
OF CHEMICALS
Compound
TESTED
Maximal
response
a,b
Purity
Source ’
BDH BCR BCR
D7 gene conversion
XV18514C
1,3-dichloroacetone l,l,l-trichloroacetone 1,1,3-trichloroacetone
+ (2X, -S9; 1.8x, +S9) + (2.5x, -s9; 1.4x, +s9) + (2.7x, -S9; 4x, +S9)
w(1.7x,Trp+, ? w(l4x,Trp+,
+S9)
Unknown 97% 98+%
1,1,1,3-tetrachloroacetone 1,1,3,3-tetrachloroacetone pentachloroacetone
w (1.9 X) -S9;
+ (2.1 X, His+,
-S9)
98+%
BCR
w(1.6x,Trp+,
+S9)
96%
Aldrich
Technical grade
Aldrich
hexachloroacetone dibromochloromethane bromodichloromethane
+ (6x, -S9; 2x, + (2.2 X) - S9) W(1.5X. -S9)
98% 98% 97%
Aldrich
trichloroacetaldehyde 3-chlorobenzyl alcohol 4-chlorobenzyl alcohol
?
99% Unknown Unknown
BDH ICN ICN
_
99% CC grade Distilled
Aldrich Aldrich Eastman
_
98%
BCR
99% Research grade
Aldrich
Unknown Unknown
ICN ICN
succinic acid methyl succinic acid diethyl succinic acid 9,10-dichloro-9,10-dihydroxystearic acid methyl dichloroacetate ethyl chloroacetate
ethyl dichloroacetate ethyl trichloroacetate
+ (2X, + (2.2X,
?
_
1.6x,
+S9)
-S9)
reversion +S9; 1.5X, His+,
-S9)
+S9)
_ w(1.9~,His+,
-S9)
+ (2.3 x , Tip+, ? _
+ S9)
_
_
-S9)
F/B Aldrich
P/B
Explanation of designations: + , positive response, defined by absolute increases in numbers of convertants or revertants on test plates, evidence for dose-related effects, and at least a 2-fold increase in mutants/survivors; w, weakly positive response, defined by absolute increases in mutants but without a dose response and with less than a doubling of mutation frequency; ?, a questionable response, marked by no absolute increases in mutant numbers but with increasing mutation frequencies with decreasing survival; - , negative response, defined by no increase in either absolute numbers or frequencies of mutants. Values in parentheses indicate the fold-increases over the solvent controls. Abbreviations for suppliers of chemicals: BDH, British Drug House; BCR, B.C. RESEARCH; F/B, A.G. Fluka/S.B. Buchs; ICN, ICN-K&K; P/B, Pfaltz and Bauer.
mutagens with yeast remains unresolved. Strain XV18514C detected 8 mutagens in the early study but failed to detect 7 bacterial mutagens in the present study (Table 3). On the other hand, none of the mutagens in the earlier study induced gene conversion in strain D7, whereas 6 of the 7 bacterial mutagens and 1 other compound were positive in D7 in this study. This comparison suggests that
the protocol for the early study may be more optimal for detection of mutagenicity in strain XV185-14C, whereas testing with D7 may be better suited to the protocol used here. Further work using the same compounds with both protocols should be done. The presence of S9 in this study did enable the detection of effects in XV185-14C but not in D7.
2
chloroacetone
1,1.1,3-tetra-
acetone
1,1,3-trichloro-
acetone
l,l,l-trichloro-
acetone
Compound
COMPOUNDS
TABLE
88(189) 87(164)
93 62
84 74
0.005
0.01
62
105 52
(0.05)
(0.1)
94 60
61
(0.005)
83(177)
98(355) 108(338)
104
103
(O.OQO5)
77(302)
89
116
111
(0.00025)
80(307)
56(197)
116(253)
152(416)
134(473)
(0.001)
118
105
109
(0.0001)
100
67
62
(0.1)
0
103
78
(0.05)
115
100
(0.01)
121(434)
118
102
(0.005)
94(343)
56(197)
85(97)
80(184)
8q202)
69(157)
117
104
(0.001)
105
116
110
100
121 119
104
(O.cnl5)
(0.01)
0
108
101
0
(0.001)
34(79) 63(140)
90(197)
103
87
0.001
105
69(193)
109
111
0.0005
105
44017) 62(176)
95
0 104
56(95)
+s9
61(92)
6q150)
61(159)
48(131)
47(140)
38(101)
158(267)
78(203)
93(272)
61(182)
59(174)
38(101)
79(81)
48(93)
44(87) 54(107)
37(67)
40(70)
98(109)
75(125)
65(119)
6q124)
59(110)
convert.
105
-s9
-s9
111
Trp+ +s9
Survival
in D7
0.0001
1”S (sl) per ml
RESPONSES
Response
POSITIVE
Dose
WITH
108 105 92 41 106 91 93 159 105 46 106 108 95 121 102 58
112 103 38 103 104 114 126 94 58 103 104 128 108 108 68
51
118
57
87
95
85
95
100
128
100
120
100
97
107
111 103
108
+s9
revert.
24(53) 25(54)
22(81)
23(45)
19(60)
13(41)
15(44) 13(46)
18(53)
27(45)
21(57)
17(63)
17(54)
19(57)
35(44)
23(51)
21(55)
26(54)
22(51)
19(43)
34(34)
27(61)
17(57)
27(55)
25(48)
19(43)
46(38)
47(56) 18(53)
28(51)
2q84) 25(82)
25(92)
24(45) 23(46)
3q28)
29(33)
22(49)
22(35)
19(45)
13(37)
+s9
27(89)
44(77)
36(108)
31(100)
31(106)
30(98)
29(106)
-s9
Trp+
in XV185-14C
-s9
Survival
Response
14(27)
18(54)
lO(32)
8.0(30)
12(36)
8.0(25)
15(25)
13(34)
8.0(30)
9.0(31)
12(35)
8.0(25)
14(45) 30(35)
16(54)
16(60)
16(60)
15(51)
16(28)
ll(33)
15(49)
lO(35)
lo(37) 14(45)
-s9
21(40)
21(23)
18(29)
15(36)
15(29)
22(28)
16(35)
12(32)
14(29)
13(30)
13(30)
28(28)
13(30)
lO(34)
17(34)
18(35)
13(30)
39(32)
21(39)
21(45)
22(47)
2.1(4)
1.3(4)
1.0(3)
0.8(3)
1.3(4)
1.7(5)
1.8(3)
l.Y5) 1.5(4)
1.5(5)
1.0(3)
1.7(5)
11.0(13)
3.1(10)
2.9(10)
2.7(10)
2.q8) 2.1(8)
4.0(7)
3.7(11)
3.7(12)
3.9(13) 3.5(12)
2.w
ww 15(30)
-s9
Horn+ +s9
25(51)
His+ revert.
1.4(3) 3.2(4)
2.0(4) 1.1(3)
1.7(4)
1.8(4)
2.0(2)
1.8(4)
1.7(6)
1.5(3)
1.0(2)
1.8(4)
6.1(5)
3.2(6)
3.3(7)
2.8(6)
3.5(7)
3.1(6)
5.5(6)
6.7(11)
2.8(7)
2.6(5)
3.0(6)
2.8(6)
+s9
revert.
trichloroacetaldehyde
dibromochloromethane
bromodichloromethane
hexachloroacetone
pentachloroacetone
1,1,3,3-tetrachloroacetone
0
0 50 100 250 500 1000
(0.1) (0.5)
0 (0.001) (0.01) (0.05)
(0.1)
0 (0.001) (0.005) (0.01) (0.05)
(0.1)
0 (0.001) (0.005) (0.01) (0.05)
(0.1)
0 (0.001) (0.005) (0.01) (0.05)
(0.0001) (0.0005) (0.001) (0.005) (0.01)
96 90 86 86 99 56
100 99 96 105 103 31 108 108 107 105 114 58
111 107 105 97 117 51
111 114 115 114 108 91
105 101 125 107 99 55
105 115 115 106 87 33 101 103 101 104 105 71
99 98 100 97 93 59
106 105 104 103 99 81
95 95 94 97 90 53
108 118 112 100 108 54
75(249) 88(276) 92(271) 93(273) 80(272) 141(222)
31(85) 36(97) 52(135) 56(161) 64(178) 84(71)
31(85) 35(96) 45(125) 31(87) 41(117) 32(62)
34(79) 37(94) 55(138) 87(201) 124(235) 201(145)
58(198) 66(223) 85(287) lOl(352) lOl(324) 128(241)
47(121) 59(166) 74(197) 74(178) 65(168) 93(121)
81(213) 79(207) 71(184) 77(197) 66(181) 113(159)
38(?4) 34(66) 33(58) 34(73) 67(62)
36(73)
44(72)
36(73) 34(71) 40( 84) 37(77) 35(69)
40(70) 36(75) 48(99) 62(110) 66(108) 78(71)
50(113) 69(154) 61(139) 57(125) 62(132) 65(88)
53(96) 53(95) 55(98) 60(107) 60(102) 54(76)
100 93 84 86 83 50
98 109 101 94 91 50
98 98 100 94 103 66
107 100 112 112 89 66
99 99 100 101 94 70
91 90 74 75 100 43
107 114 112 107 102 72
97 116 103 97 104 80
97 177 164 172 104 86
95 86 101 92 83 64
98 102 106 109 106 92
103 128 89 122 97 65
20(67) 22(69) 22(64) 21(62) 27(77) 36(61)
12(32) 15(42) 12(32) 15(36) 13(32) 13(17)
12(32) 7.0(18) 8.0(22) 13(31) 13(36) lO(17)
27( 89) 24(75) 22(76) 32(112) 38(105) 38(78)
29(82) 28(80) 26(75) 27(79) 28(77) 36(74)
32(90) 27(75) 44(100) 40(92) 29(87) 25(33)
2.4(4)
2.4(7) 2.7(8) 3.9(11)
17(40) 13(30) 16(36) 18(25) 14(39) 15(45) ll(32) 15(25) 18(44) 20(47) 24(53) 42(64)
2.4(8) 2.9(9)
1.2(3) 0.7(2) 1.1(3) 1.2(3) 0.8(2) 0.0(O)
1.0(3) 1.0(2) 1.0(2) 1.0(3) 1.0(3) 0.6(l)
2.6(9). 2.9(8) 2.4(5)
2.4(8) 3.2(10) 2.9(10)
2.4(7) 2.2(6) 2.0(4)
3.8(11) 2.4(7) 3.1(9)
2.9(8) 4.4(12) 5.3(12) 6.5(15) 3.3(10) 4.6(6)
13(31) 15(36)
8.0(12) 9.0(16) 9.0(14) 8.0(13) 7.0(12) 2.0(2)
8.0(12) 5.0(15) 7.0(18) 6.0(15) lO(16) 7.0(10)
21(40) 21(37) 19(38) 21(38) 20(33) 18(23)
18(33) 17(32) 16(32) 18(37) 17(34) 12(20)
12(28) lO(29) 17(33) 12(32) 17(36) 17(24)
9.0(31) 12(38)
9.0(22) ll(31) 8.0(22) 8.0(20) 8.0(19) 6.0(8)
9.0(22) ll(29) 15(40) 17(41) 13(34) 13(22)
15(51) 20(63) 18(64) 16(57) 19(52) 21(43)
16(45) 14(41) 13139) 13(37) 17(46) 9.0(18)
13(35) 13(36) 16(36) 18(41) 12(37) 18(23)
18(42) 18(45)
14(21) 13(21) ll(14)
12(19) 12(21) 13(21)
12(19) 7.0(19) 8.0(22) 6.0( 17) 12(19) lO(13)
24(45) 35(60) 23(46) 32(59) 36(60) 28(36)
2q45) 30(60) 25(31) 25(50) 24(42)
26(47)
31(44)
14(32) 20(55) 28(54) 29(76) 42(89)
2.4(6) 2.5(6) 2.2(5) 2.7(6) 2.6(4)
2.2(5)
2.4(4) 0.0(O)
1.3(2) 3.0(5) 4.0(6) 3.0(4)
1.0(2) 1.0(4) 1.O(3) 1.0(3) 1.0(2) 1.5(2)
3.2(6) 4.0( 7) 4.4(9) 5.4(10) 3.6(6) 3.9(5)
3.8(7) 5.3(10) 5.0(10) 3.9(8) 5.5(11) 5.8(10)
3.6(8) 2.5(7) 3.1(6) 2.7(7) 2.8(6) 4.9(7)
Dose
93106
(0.5)
1.0
ethyl methanesulfonate
2-aminoanthracene 119
105.
100
85(172) 132(354)
36(66)77(188)
30(80)69(242) 187(607)311(722)
56(118) 55(118) 47(120) 45(115) 58(125) 112(112)
44054) SO(169) Sl(l64) 56(170) 84(113)
85 102
100
89116
100
103 104 100 103 82 42
104 108 104 80 38
101
46(161)
103 105 124 125 108 49
9s 101 98 93 88 39
100
0 0.1 1.0 2.5 5.0 10.0
4-chlorobenzyl alcohol
44(96) 4qlOO) SO(102) 62(115) 65(67)
56(118)
48(160) 49(162) 53(154) 58(167) 95(124) 91(73)
103 107 111 99 90 50
95 96 84 83 37 23
+s9 103 93 96 101 59 23
-s9
-s9
revert.
130(357)262(764)
13(35)30(101)
34(116) 34(116) 29(106) 29(102) 38(103) 68(86)
34(116) 29(98) 27(92) 29(99) 57(94) 158(95)
-s9
Trp+
in XV185-14C
101 102 102 102 so 18
Survival +s9
Trp + convert. +s9
Response
-s9
in D7
Survival
Response
negative control (nothing added)
0 (0.001) (0.0025) (0.005) (0.01) (0.05)
!Jg (al) per ml
3-chlorobenzyl alcohol
Compound
TABLE 2 (continued)
37(82)55(121)
11(18)26(56)
27(64) 24(58) 27(64) 28(67) 34(65) 51(49)
26(64) 25(55) 29(65) 27(63) 41(57) 115(62)
+s9
97(267)209(622)
8.0(20)18(57)
lO(34) lO(34) 14(49) 12(47) 13(35) 25(31)
lo(34) ll(37) ll(36) 21(35) 63(38)
lO(34)
-s9
His+ revert.
30(68)55(99)
8.0(13)19(39)
lq32) 13(31) 20(38) 28(27)
12(29) lS(37)
lq33) 21(29) 41(22)
12(29) 13(28) 17(37)
+s9
7.5(19)56(108)
1.0(3)3.3(10)
2.4(3)
l.q5) 1.4(S) M(4) 2.2(6)
1.8(6)
9.2(19)19 (38)
1 .q3)4.8(9)
5.4(S) 4.1(4)
1.2(3) 1.6(4) 1.7(4) 0.8(2)
+s9 1.2(3) 3.7(8) 3.6(8) 2.1(S) 2.9(4) 7.4(4)
1.8(6) 1.8(6) 1.8(6) 1.8(6) 4.8(8) 8.3(5)
revert. -s9
Horn+
59
TABLE
3
SUMMARY
OF TEST RESULTS
IN YEAST AND BACTERIA S. cereuisae
Compound
Salmonella
Gene conversion
Reverse mutation
Reverse mutation
Reference
1,3-dichloroacetone 1,1,3-trichloroacetone 1,1,3,3-tetrachloroacetone
+ + +
w w w
+ + +
Kringstad et al., 1981 Douglas et al., 1984 ibid.
pentachloroacetone hexachloroacetone dibromochloromethane
+ + +
_ _ _
+ + +a
ibid. ibid. Simmon et al., 1977
bromodichloromethane trichloroacetaldehyde
w ?
w +
+a _b
ethyl chloroacetate ethyltrichloroacetate 3- or 4-chlorobenzyl alcohol succinic acid methyl succinic acid
_ _ _ _
_ _ _
_b _b _c _c _c
ibid. Kringstad et al., 1981 ibid. ibid. Douglas et al., 1984 ibid. ibid.
a A desiccator assay was used. b In strain TA1535 only. ’ In strains TA98 and TAlOO only.
TABLE
4
DIFFERENCES Nestmann
IN TEST PROCEDURES
and Lee (1983)
This study
(1) Cell concentrations lower (cells not suspended with reciprocal bath). (2) Mutant backgrounds variable. (3) Treatment for 24 h (4) Cells plated without washing. (5) No S9 used.
Acknowledgements We wish to thank Potter for their manuscript, and C. patient and untiring script for publication.
Drs. A.F. Hanham and A.A. comments concerning this Gilchrist and K. Nesbitt for efforts in preparing the type-
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31, 347-363.
(1) Cell concentrations higher with gyratory shaker bath. (2) Stock cultures chosen for low spontaneous backgrounds. (3) Treatment for 16 h. (4) Cells chilled and washed before before plating. (5) Treatments f S9.
Ander, P., K.-E. Eriksson, M.-C. Kolar, K. Kringstad, U. Rannug and C. Ramel(1977) Studies on mutagenic properties of bleaching effluents, Part I, Svensk Papperstidn., 80, 454-459. Bjsrseth, A., G.E. Carlberg and M. Moller (1979) Determination of halogenated organic compounds and mutagenicity testing of spent bleach liquors, Sci. Total Environ., 11, 197-211. de Serres, F.J., and G.R. Hoffmann (1981) Summary report on the performance of yeast assays, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 68-76. Douglas, G.R., E.R. Nestmann, J.L., Betts, J.C. Mueller, E.G.H. Lee, H.F. Stich, R.H.C. San, R.P. Brouzes, A.L. Chmelauskas, H.D. Paavila and CC. Walden (1980) Mutagenic activity in pulp mill effuents, in: R.L Jolley, W.A.
60
Brungs and R.B. Cumming (Eds.), Water Chlorination: Environmental Impact and Health Effects, Vol. 3, Ann Arbor Science, Ann Arbor, pp. 865-880. Douglas, G.R., E.R. Nestmann, A.B. McKague, O.P. Kamra, E.G.-H. Lee, J.A. Ellenton, R. Bell, D. Kowbel, V. Liu and J. Pooley (1983) Mutagenicity of pulp and paper mill effluent: a comprehensive study of complex mixtures, in: M. Waters, S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff and S. Nesnow (Eds.), Application of Short-Term Bioassays in the Analysis of Complex Environmental Mixtures, Vol. 3, Plenum, New York, pp. 431-459. Douglas, G.R., E.R. Nestmann, A.B. McKague, R.H.C. San, E.G.-H. Lee, V.W. Liu-Lee and D.J. Kowbel (1984) Determination of potential hazard from pulp and paper mills: mutagenicity and chemical analysis, in: H.F. Stich (Ed.), Carcinogens and Mutagens in the Environment, Vol. IV, The Workplace, CRC Press, Boca Raton, in press. Ellenton, J.A., G.R. Douglas and E.R. Nestmann (1981) Mutagenic evaluation of 1,1,2,3-tetrachloropropene, a contaminant in pulp mill effluents, using a battery of in vitro mammalian and microbial tests, Can. J. Genet. Cytol., 23, 17-25. Eriksson, K.-E., M.-C. Kolar and K. Kringstad (1979) Studies on mutagenic properties of bleaching effluents, Part 2, Svensk Papperstidn., 82, 95-104. Hannan, M.A. and A. Nasim (1978) Genetic activity of bleomycin: Differential effects on mitotic recombination and mutations in yeast, Mutation Res., 53, 309-316. Kamra, O.P., E.R. Nestmann, G.R. Douglas, D.J. Kowbel and T.R. Harrington (1983) Genotoxic activity of pulp mill effluent in Salmonella and Saccharomyces cerevisiae assays, Mutation Res., 118, 269-276. Kringstad, K.P., P.O. Ljungquist, F. de Sousa and L.M. Stromberg (1981) Identification and mutagenic properties of some chlorinated aliphatic compounds in the spent liquor from kraft pulp chlorination, Environ. Sci. Technol., 15, 562-566. Mehta, R.D., and R.C. von Borstel (1981) Mutagenic activity of 42 encoded compounds in the haploid yeast reversion assay, strain XV185-14C, in: F.J. de Serres and J. Ashby @is.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 414-423. Nestmann, E.R., and G.R. Douglas (1981) New tools for assessing mutagenic and carcinogenic risk of municipal and industrial waste waters, in: M. Moo-Young and C.W.
Robinson (Eds.), Advances in Biotechnology, Vol. II, Pergamon, London, pp. 649-658, Nestmann, E.R., and E.G.-H. Lee (1983) Mutagenicity of constituents of pulp and paper mill effluent in growing cells of Saccharomyces cerevisiae, Mutation Res., 119, 273-280. Nestmann, E.R., E.G.-H. Lee, J.C. Mueller and G.R. Douglas (1979) Mutagenicity of resin acids identified in pulp and paper mill effluents using the Salmonella/mammalian-microsome assay, Environ. Mutagen., 1, 361-369. Nestmann, E.R., E.G.-H. Lee, T.I. Matula, G.R. Douglas and J.C. Mueller (1980) Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/mammalian-microsome assay, Mutation Res., 79, 203-212. Nestmann, E.R., A. Nasim, R.H. Haynes and D.J. Kowbel (1981) Genetic activity of actinomycin D in Saccharomyces cerevisiae but not in kcherichia coli, Mutation Res., 89, 229-236. Parry, J.M. (1985) Summary Report on the performance of the yeast and Aspergillus assays, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Elsevier, Amsterdam, pp. 25-46. Rapson, W.H., M.A. Nazar and V.V. Butsky (1980) Mutagenicity produced by aqueous chlorination of organic compounds, Bull. Environ. Contam. Toxicol., 24, 590-596. Simmon, V.F. (1979) In vitro assays for recombinogenic activity of chemical carcinogens and related compounds with Saccharomyces cerevisiae D3, J. Natl. Cancer Inst., 72, 901-909. Simmon, V.F., K. Kauhanen and R.G. Tardiff (1977) Mutagenic activity of chemicals identified in drinking water, in: D. Scott, B.A. Bridges and F.H. Sobels (Eds.), Progress in Genetic Toxicology, Developments in Toxicology and Environmental Science, Vol. 2, Elsevier, Amsterdam, pp. 249-258. Von Borstel, R.C., M.M. Shahin and R.D. Mehta (1981) Protocol for a haploid yeast reversion test for assaying mutagens, in: H.F. Stich and R.H.C. San (Eds.), Short-Term Tests for Chemical Carcinogens, Springer, New York, pp. 171-174. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for simultaneous detection of induced crossing over, mitotic gene conversion, and reverse mutation, Mutation Res., 28, 381-388.
53
MTR 00952
Genetic activity in Saccharomyces cereuisiae of compounds effluents of pulp and paper mills Earle R. Nestmann a Mutagenesis
found in
a* and Ernie G.-H. Lee b
Section, Environmental and Occupational Toxicology Division, Deparlment of National Health and Welfare, Ottawa, Ontario KlA OL2 and b B.C. Research, 3650 Wesbrook Mall, Vancouver, BC, V6S 2L2 (Canada) (Received (Accepted
11 September 14 September
1984) 1984)
Summary 20 compounds
in pulp mill effluents were screened for genetic activity in growing cells using D7 and XV185-14C without and with S9. Nine compounds were positive in one or the other yeast strain (7 in D7; 2 in XV18514C). One additional compound showed weak effects and two others showed elevated frequencies/survivor without absolute increases of mutants. The presence of S9 enabled detection of one positive and two weak effects, it enhanced the genetic activity of one compound in each strain, and it reduced the mutagenic effects of 4 others in strain D7. 7 of the 20 chemicals tested have been shown previously to be mutagenic in the Salmonella/mammalian-microsome assay. Of the 7 bacterial mutagens, 6 were positive and 1 had a weak effect in yeast. Saccharomyces
identified
cerevisiae strains
Recent international collaborative studies have shown that yeast assays are not only promising predictors of carcinogenic activity (de Serres and Hoffmann, 1981), but that they compare favorably in performance with other eukaryotic test systems in correctly identifying selected chemical carcinogens (Parry, 1985). The literature contains an increasing number of examples of mutagens that were detected in yeast but not in prokaryotes (Hannan and Nasim, 1978; Simmon, 1979; Nestmann et al., 1981; Nestmann and Lee, 1983), leading many laboratories of genetic toxicology to adopt Saccharomyces cerevisiae as a useful test. The present study continues our efforts to characterize the genetic toxicity associated with ef-
* Correspondence and reprint requests should be addressed to Dr. Earle R. Nestmann, Environmental Health Centre, Tunney’s Pasture, Ottawa KlA 0L2 (Canada). 0165-1218/85/$03.30
0 1985 Elsevier Science Publishers
fluents from pulp and paper mills. Such environmental mixtures are studied with different approaches; for example, by testing the complex mixture (or extracts from it) or by assessing its constituent compounds individually (Nestmann and Douglas, 1981). Previous reports have shown that effluent from pulp and paper mills induces genetic effects in a variety of test organisms (Ander et al., 1977; Eriksson et al., 1979; Douglas et al., 1980, 1983, 1984), including yeast (Kamra et al., 1983). Another strategy to identify compounds that contribute to the mutagenicity of the mixture is its sequential sub-fractionation using mutagenic activity as a means of following the active fractions (i.e., mutagenicity-directed fractionation; Douglas et al., 1984). This approach may be misleading, due to many possible interactions among the mutagens and non-mutagens present, or difficult, if mixtures contain a large number of mutagens. Many compounds in pulp and paper
B.V. (Biomedical
Division)
54
mill effluent have been shown to be mutagenic in Salmonella (Bjorseth et al., 1979; Nestmann et al., 1979, 1980; Rapson et al., 1980; Douglas et al., 1983), in yeast (Nestmann and Lee, 1983), and in mammalian cells (Ellenton et al., 1981; Douglas et al., 1983). In the present study, 20 compounds were assessed for reverse mutation and gene conversion in growing cultures of yeast. A preliminary report of this study was presented at the 11th International Conference on Yeast Genetics and Molecular Biology in Montpellier, France (September 1982). Yeast strains D7 (Zimmermann et al., 1975) and XV18514C (von Borstel et al., 1981) were used for testing according to the method described by von Borstel et al. (1981) using exponential phase cultures for Trp+ gene convertants or for reversion of the histidine, homoserine and tryptophan markers, respectively. In XV18514C, the trpS-48 mutation is ochre-suppressible and, therefore, can be reverted by mutations either within the gene or in a suppressor; the his1 -7 marker is a missense mutation and is relatively sensitive since it reverts largely at secondary sites; and there is evidence that the hom3-IO mutation is reverted by frameshift mutations (Mehta and von Borstel, 1981). Cells from frozen YEPD cultures (- SO’C) that were selected after preliminary screening for low mutation frequencies, were streaked on YEPD agar plates. After 2 days (3O”Q liquid subcultures (50 ml) were grown for 16 h in a gyratory water bath (30°C). For the tests, these growing cells were centrifuged and resuspended in YEPD (1 X 10’ cells/ml) with 25% of either phosphate buffer or S9 mix. Samples (up to 25 ~1) of solvent or test compound were combined with 2 ml of the cell suspension (+ S9) in a screw-top tube (16 X 125 mm) and incubated for 16 h either in a 30°C gyratory water bath ( - S9) or in a 37’C reciprocal water bath (+ S9). Treated cultures were chilled, washed, diluted 2-fold in phosphate buffer, and plated (0.2 ml/plate) on omission media for convertants and revertants. Further dilutions (10’) were made for survival determinations. All platings were made in triplicate, and the plates were incubated for 4 days (2 days for survival). Each experiment included 2 negative and 2 positive controls: nothing added to the culture and solvent;
ethyl methanesulfonate and 2-aminoanthracene (for S9). A response was considered to be positive when the numbers of mutants were higher than on the control plates and when the calculated mutant frequency (per lo5 survivors) was at least double the solvent control (see legend to Table 1). Dichloroacetone, succinic acid and methyl succinic acid were dissolved in water. The other 17 compounds were either diluted or dissolved in dimethyl sulfoxide (DMSO). Table 1 is a summary of the positive, weakly positive, questionable, and negative responses found. 7 compounds were found to induce Trp+ gene convertants in strain D7. None of these 7 was considered positive at any of 3 loci in haploid strain XV185-14C, although weak effects were found. 2 other chemicals induced His’ or Trp+ revertants in strain XV185-14C. As complementary yeast tests, these yeast strains detected a total of 9 positive compounds. 1 compound (bromodichloromethane) induced weak effects in both tests. 2 others (3- and 4-chloro-benzyl alcohol), showed higher frequencies of convertants or revertants per survivor without increases in numbers of mutant colonies per plates. Data for these positive, weak and questionable responses are given in Table 2. Of the 9 compounds found to have genetic activity, 6 have been been reported previously to be positive in the Salmonella assay, 1 was negative (in strain TA1535 only), and 2 are untested. 1 compound that had weak effects in yeast was positive in bacteria only when tested in a desiccator as an exposure chamber. Negative results for both Saccharomyces and Salmonella were observed for 5 compounds. Details of these comparisons and references are shown in Table 3. In comparison with a previous study of the genetic activity of 42 compounds in the same yeast strains (Nestmann and Lee, 1983) a much larger proportion of mutagens (9 of 20, or 45%) were found in this study than before (8 of 42, or 19%). However, the choices of compounds for each study were not random. Previous studies of these groups of compounds in Salmonella showed different proportions of bacterial mutagens, 35 and 7% respectively. The experimental protocols differed slightly in the two yeast studies (Table 4), and the question of what protocol is optimal in screening for
55 TABLE
1
SUMMARY
OF CHEMICALS
Compound
TESTED
Maximal
response
a,b
Purity
Source ’
BDH BCR BCR
D7 gene conversion
XV18514C
1,3-dichloroacetone l,l,l-trichloroacetone 1,1,3-trichloroacetone
+ (2X, -S9; 1.8x, +S9) + (2.5x, -s9; 1.4x, +s9) + (2.7x, -S9; 4x, +S9)
w(1.7x,Trp+, ? w(l4x,Trp+,
+S9)
Unknown 97% 98+%
1,1,1,3-tetrachloroacetone 1,1,3,3-tetrachloroacetone pentachloroacetone
w (1.9 X) -S9;
+ (2.1 X, His+,
-S9)
98+%
BCR
w(1.6x,Trp+,
+S9)
96%
Aldrich
Technical grade
Aldrich
hexachloroacetone dibromochloromethane bromodichloromethane
+ (6x, -S9; 2x, + (2.2 X) - S9) W(1.5X. -S9)
98% 98% 97%
Aldrich
trichloroacetaldehyde 3-chlorobenzyl alcohol 4-chlorobenzyl alcohol
?
99% Unknown Unknown
BDH ICN ICN
_
99% CC grade Distilled
Aldrich Aldrich Eastman
_
98%
BCR
99% Research grade
Aldrich
Unknown Unknown
ICN ICN
succinic acid methyl succinic acid diethyl succinic acid 9,10-dichloro-9,10-dihydroxystearic acid methyl dichloroacetate ethyl chloroacetate
ethyl dichloroacetate ethyl trichloroacetate
+ (2X, + (2.2X,
?
_
1.6x,
+S9)
-S9)
reversion +S9; 1.5X, His+,
-S9)
+S9)
_ w(1.9~,His+,
-S9)
+ (2.3 x , Tip+, ? _
+ S9)
_
_
-S9)
F/B Aldrich
P/B
Explanation of designations: + , positive response, defined by absolute increases in numbers of convertants or revertants on test plates, evidence for dose-related effects, and at least a 2-fold increase in mutants/survivors; w, weakly positive response, defined by absolute increases in mutants but without a dose response and with less than a doubling of mutation frequency; ?, a questionable response, marked by no absolute increases in mutant numbers but with increasing mutation frequencies with decreasing survival; - , negative response, defined by no increase in either absolute numbers or frequencies of mutants. Values in parentheses indicate the fold-increases over the solvent controls. Abbreviations for suppliers of chemicals: BDH, British Drug House; BCR, B.C. RESEARCH; F/B, A.G. Fluka/S.B. Buchs; ICN, ICN-K&K; P/B, Pfaltz and Bauer.
mutagens with yeast remains unresolved. Strain XV18514C detected 8 mutagens in the early study but failed to detect 7 bacterial mutagens in the present study (Table 3). On the other hand, none of the mutagens in the earlier study induced gene conversion in strain D7, whereas 6 of the 7 bacterial mutagens and 1 other compound were positive in D7 in this study. This comparison suggests that
the protocol for the early study may be more optimal for detection of mutagenicity in strain XV185-14C, whereas testing with D7 may be better suited to the protocol used here. Further work using the same compounds with both protocols should be done. The presence of S9 in this study did enable the detection of effects in XV185-14C but not in D7.
2
chloroacetone
1,1.1,3-tetra-
acetone
1,1,3-trichloro-
acetone
l,l,l-trichloro-
acetone
Compound
COMPOUNDS
TABLE
88(189) 87(164)
93 62
84 74
0.005
0.01
62
105 52
(0.05)
(0.1)
94 60
61
(0.005)
83(177)
98(355) 108(338)
104
103
(O.OQO5)
77(302)
89
116
111
(0.00025)
80(307)
56(197)
116(253)
152(416)
134(473)
(0.001)
118
105
109
(0.0001)
100
67
62
(0.1)
0
103
78
(0.05)
115
100
(0.01)
121(434)
118
102
(0.005)
94(343)
56(197)
85(97)
80(184)
8q202)
69(157)
117
104
(0.001)
105
116
110
100
121 119
104
(O.cnl5)
(0.01)
0
108
101
0
(0.001)
34(79) 63(140)
90(197)
103
87
0.001
105
69(193)
109
111
0.0005
105
44017) 62(176)
95
0 104
56(95)
+s9
61(92)
6q150)
61(159)
48(131)
47(140)
38(101)
158(267)
78(203)
93(272)
61(182)
59(174)
38(101)
79(81)
48(93)
44(87) 54(107)
37(67)
40(70)
98(109)
75(125)
65(119)
6q124)
59(110)
convert.
105
-s9
-s9
111
Trp+ +s9
Survival
in D7
0.0001
1”S (sl) per ml
RESPONSES
Response
POSITIVE
Dose
WITH
108 105 92 41 106 91 93 159 105 46 106 108 95 121 102 58
112 103 38 103 104 114 126 94 58 103 104 128 108 108 68
51
118
57
87
95
85
95
100
128
100
120
100
97
107
111 103
108
+s9
revert.
24(53) 25(54)
22(81)
23(45)
19(60)
13(41)
15(44) 13(46)
18(53)
27(45)
21(57)
17(63)
17(54)
19(57)
35(44)
23(51)
21(55)
26(54)
22(51)
19(43)
34(34)
27(61)
17(57)
27(55)
25(48)
19(43)
46(38)
47(56) 18(53)
28(51)
2q84) 25(82)
25(92)
24(45) 23(46)
3q28)
29(33)
22(49)
22(35)
19(45)
13(37)
+s9
27(89)
44(77)
36(108)
31(100)
31(106)
30(98)
29(106)
-s9
Trp+
in XV185-14C
-s9
Survival
Response
14(27)
18(54)
lO(32)
8.0(30)
12(36)
8.0(25)
15(25)
13(34)
8.0(30)
9.0(31)
12(35)
8.0(25)
14(45) 30(35)
16(54)
16(60)
16(60)
15(51)
16(28)
ll(33)
15(49)
lO(35)
lo(37) 14(45)
-s9
21(40)
21(23)
18(29)
15(36)
15(29)
22(28)
16(35)
12(32)
14(29)
13(30)
13(30)
28(28)
13(30)
lO(34)
17(34)
18(35)
13(30)
39(32)
21(39)
21(45)
22(47)
2.1(4)
1.3(4)
1.0(3)
0.8(3)
1.3(4)
1.7(5)
1.8(3)
l.Y5) 1.5(4)
1.5(5)
1.0(3)
1.7(5)
11.0(13)
3.1(10)
2.9(10)
2.7(10)
2.q8) 2.1(8)
4.0(7)
3.7(11)
3.7(12)
3.9(13) 3.5(12)
2.w
ww 15(30)
-s9
Horn+ +s9
25(51)
His+ revert.
1.4(3) 3.2(4)
2.0(4) 1.1(3)
1.7(4)
1.8(4)
2.0(2)
1.8(4)
1.7(6)
1.5(3)
1.0(2)
1.8(4)
6.1(5)
3.2(6)
3.3(7)
2.8(6)
3.5(7)
3.1(6)
5.5(6)
6.7(11)
2.8(7)
2.6(5)
3.0(6)
2.8(6)
+s9
revert.
trichloroacetaldehyde
dibromochloromethane
bromodichloromethane
hexachloroacetone
pentachloroacetone
1,1,3,3-tetrachloroacetone
0
0 50 100 250 500 1000
(0.1) (0.5)
0 (0.001) (0.01) (0.05)
(0.1)
0 (0.001) (0.005) (0.01) (0.05)
(0.1)
0 (0.001) (0.005) (0.01) (0.05)
(0.1)
0 (0.001) (0.005) (0.01) (0.05)
(0.0001) (0.0005) (0.001) (0.005) (0.01)
96 90 86 86 99 56
100 99 96 105 103 31 108 108 107 105 114 58
111 107 105 97 117 51
111 114 115 114 108 91
105 101 125 107 99 55
105 115 115 106 87 33 101 103 101 104 105 71
99 98 100 97 93 59
106 105 104 103 99 81
95 95 94 97 90 53
108 118 112 100 108 54
75(249) 88(276) 92(271) 93(273) 80(272) 141(222)
31(85) 36(97) 52(135) 56(161) 64(178) 84(71)
31(85) 35(96) 45(125) 31(87) 41(117) 32(62)
34(79) 37(94) 55(138) 87(201) 124(235) 201(145)
58(198) 66(223) 85(287) lOl(352) lOl(324) 128(241)
47(121) 59(166) 74(197) 74(178) 65(168) 93(121)
81(213) 79(207) 71(184) 77(197) 66(181) 113(159)
38(?4) 34(66) 33(58) 34(73) 67(62)
36(73)
44(72)
36(73) 34(71) 40( 84) 37(77) 35(69)
40(70) 36(75) 48(99) 62(110) 66(108) 78(71)
50(113) 69(154) 61(139) 57(125) 62(132) 65(88)
53(96) 53(95) 55(98) 60(107) 60(102) 54(76)
100 93 84 86 83 50
98 109 101 94 91 50
98 98 100 94 103 66
107 100 112 112 89 66
99 99 100 101 94 70
91 90 74 75 100 43
107 114 112 107 102 72
97 116 103 97 104 80
97 177 164 172 104 86
95 86 101 92 83 64
98 102 106 109 106 92
103 128 89 122 97 65
20(67) 22(69) 22(64) 21(62) 27(77) 36(61)
12(32) 15(42) 12(32) 15(36) 13(32) 13(17)
12(32) 7.0(18) 8.0(22) 13(31) 13(36) lO(17)
27( 89) 24(75) 22(76) 32(112) 38(105) 38(78)
29(82) 28(80) 26(75) 27(79) 28(77) 36(74)
32(90) 27(75) 44(100) 40(92) 29(87) 25(33)
2.4(4)
2.4(7) 2.7(8) 3.9(11)
17(40) 13(30) 16(36) 18(25) 14(39) 15(45) ll(32) 15(25) 18(44) 20(47) 24(53) 42(64)
2.4(8) 2.9(9)
1.2(3) 0.7(2) 1.1(3) 1.2(3) 0.8(2) 0.0(O)
1.0(3) 1.0(2) 1.0(2) 1.0(3) 1.0(3) 0.6(l)
2.6(9). 2.9(8) 2.4(5)
2.4(8) 3.2(10) 2.9(10)
2.4(7) 2.2(6) 2.0(4)
3.8(11) 2.4(7) 3.1(9)
2.9(8) 4.4(12) 5.3(12) 6.5(15) 3.3(10) 4.6(6)
13(31) 15(36)
8.0(12) 9.0(16) 9.0(14) 8.0(13) 7.0(12) 2.0(2)
8.0(12) 5.0(15) 7.0(18) 6.0(15) lO(16) 7.0(10)
21(40) 21(37) 19(38) 21(38) 20(33) 18(23)
18(33) 17(32) 16(32) 18(37) 17(34) 12(20)
12(28) lO(29) 17(33) 12(32) 17(36) 17(24)
9.0(31) 12(38)
9.0(22) ll(31) 8.0(22) 8.0(20) 8.0(19) 6.0(8)
9.0(22) ll(29) 15(40) 17(41) 13(34) 13(22)
15(51) 20(63) 18(64) 16(57) 19(52) 21(43)
16(45) 14(41) 13139) 13(37) 17(46) 9.0(18)
13(35) 13(36) 16(36) 18(41) 12(37) 18(23)
18(42) 18(45)
14(21) 13(21) ll(14)
12(19) 12(21) 13(21)
12(19) 7.0(19) 8.0(22) 6.0( 17) 12(19) lO(13)
24(45) 35(60) 23(46) 32(59) 36(60) 28(36)
2q45) 30(60) 25(31) 25(50) 24(42)
26(47)
31(44)
14(32) 20(55) 28(54) 29(76) 42(89)
2.4(6) 2.5(6) 2.2(5) 2.7(6) 2.6(4)
2.2(5)
2.4(4) 0.0(O)
1.3(2) 3.0(5) 4.0(6) 3.0(4)
1.0(2) 1.0(4) 1.O(3) 1.0(3) 1.0(2) 1.5(2)
3.2(6) 4.0( 7) 4.4(9) 5.4(10) 3.6(6) 3.9(5)
3.8(7) 5.3(10) 5.0(10) 3.9(8) 5.5(11) 5.8(10)
3.6(8) 2.5(7) 3.1(6) 2.7(7) 2.8(6) 4.9(7)
Dose
93106
(0.5)
1.0
ethyl methanesulfonate
2-aminoanthracene 119
105.
100
85(172) 132(354)
36(66)77(188)
30(80)69(242) 187(607)311(722)
56(118) 55(118) 47(120) 45(115) 58(125) 112(112)
44054) SO(169) Sl(l64) 56(170) 84(113)
85 102
100
89116
100
103 104 100 103 82 42
104 108 104 80 38
101
46(161)
103 105 124 125 108 49
9s 101 98 93 88 39
100
0 0.1 1.0 2.5 5.0 10.0
4-chlorobenzyl alcohol
44(96) 4qlOO) SO(102) 62(115) 65(67)
56(118)
48(160) 49(162) 53(154) 58(167) 95(124) 91(73)
103 107 111 99 90 50
95 96 84 83 37 23
+s9 103 93 96 101 59 23
-s9
-s9
revert.
130(357)262(764)
13(35)30(101)
34(116) 34(116) 29(106) 29(102) 38(103) 68(86)
34(116) 29(98) 27(92) 29(99) 57(94) 158(95)
-s9
Trp+
in XV185-14C
101 102 102 102 so 18
Survival +s9
Trp + convert. +s9
Response
-s9
in D7
Survival
Response
negative control (nothing added)
0 (0.001) (0.0025) (0.005) (0.01) (0.05)
!Jg (al) per ml
3-chlorobenzyl alcohol
Compound
TABLE 2 (continued)
37(82)55(121)
11(18)26(56)
27(64) 24(58) 27(64) 28(67) 34(65) 51(49)
26(64) 25(55) 29(65) 27(63) 41(57) 115(62)
+s9
97(267)209(622)
8.0(20)18(57)
lO(34) lO(34) 14(49) 12(47) 13(35) 25(31)
lo(34) ll(37) ll(36) 21(35) 63(38)
lO(34)
-s9
His+ revert.
30(68)55(99)
8.0(13)19(39)
lq32) 13(31) 20(38) 28(27)
12(29) lS(37)
lq33) 21(29) 41(22)
12(29) 13(28) 17(37)
+s9
7.5(19)56(108)
1.0(3)3.3(10)
2.4(3)
l.q5) 1.4(S) M(4) 2.2(6)
1.8(6)
9.2(19)19 (38)
1 .q3)4.8(9)
5.4(S) 4.1(4)
1.2(3) 1.6(4) 1.7(4) 0.8(2)
+s9 1.2(3) 3.7(8) 3.6(8) 2.1(S) 2.9(4) 7.4(4)
1.8(6) 1.8(6) 1.8(6) 1.8(6) 4.8(8) 8.3(5)
revert. -s9
Horn+
59
TABLE
3
SUMMARY
OF TEST RESULTS
IN YEAST AND BACTERIA S. cereuisae
Compound
Salmonella
Gene conversion
Reverse mutation
Reverse mutation
Reference
1,3-dichloroacetone 1,1,3-trichloroacetone 1,1,3,3-tetrachloroacetone
+ + +
w w w
+ + +
Kringstad et al., 1981 Douglas et al., 1984 ibid.
pentachloroacetone hexachloroacetone dibromochloromethane
+ + +
_ _ _
+ + +a
ibid. ibid. Simmon et al., 1977
bromodichloromethane trichloroacetaldehyde
w ?
w +
+a _b
ethyl chloroacetate ethyltrichloroacetate 3- or 4-chlorobenzyl alcohol succinic acid methyl succinic acid
_ _ _ _
_ _ _
_b _b _c _c _c
ibid. Kringstad et al., 1981 ibid. ibid. Douglas et al., 1984 ibid. ibid.
a A desiccator assay was used. b In strain TA1535 only. ’ In strains TA98 and TAlOO only.
TABLE
4
DIFFERENCES Nestmann
IN TEST PROCEDURES
and Lee (1983)
This study
(1) Cell concentrations lower (cells not suspended with reciprocal bath). (2) Mutant backgrounds variable. (3) Treatment for 24 h (4) Cells plated without washing. (5) No S9 used.
Acknowledgements We wish to thank Potter for their manuscript, and C. patient and untiring script for publication.
Drs. A.F. Hanham and A.A. comments concerning this Gilchrist and K. Nesbitt for efforts in preparing the type-
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31, 347-363.
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