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microsome liquid-preincubation procedure
Mutation Research, 204 (1988) 123-129 Elsevier
123
MTR 01244
Mutagenicity of methyl isocyanate in the modified test conditions of Ames Salmonella/microsome liquid-preincubation procedure Ghansham P. Meshram and Kola M. Rao Division of Microbiology, Defence Research and Development Establishment, Tansen Road, Gwalior-474 002 (India) (Received 20 February 1987) (Revision received 11 August 1987) (Accepted 25 August 1987)
Keywords: Methyl isocyanate; Modified test conditions; Ames Salmonella/microsome liquid-preincubation procedure.
Summary Methyl isocyanate (MIC) was tested for mutagenicity using the Ames Salmonella/microsome liquidpreincubation procedure with slight modification of test conditions. In the modification the preincubation mixture was incubated at 10°C for 60 min. MIC was assayed both in the presence and absence of Aroclor-1254-induced $9, using 5 tester strains of Salmonella typhimurium, TA97a, TA98, TA100, TA102 and TA104. MIC induced mutagenic response in two base-pair substitution strains, TA100 and TA104, in the presence and absence of $9. However, mutagenic response in the presence of $9 was low as compared to that in the absence of $9. In the comparative mutagenic activity at 3 different preincubation test conditions (37 °C for 20 rain, 20 °C for 40 min and 10 °C for 60 min), optimum mutagenic response was observed at 10 °C for the 60-min test condition. However, no mutagenic response was observed at 37 °C for the 20-rain test condition.
Methyl isocyanate (MIC) is an industrial chemical used as a basic ingredient in the production of various pesticides such as aldicarb, carbaryl, carbofuran and methomyl (Anon, 1984). MIC is highly irritating and volatile. It has low boiling point (39.1 ° C) and high vapour pressure (348 mm Hg at 20 o C) and consequently, it vaporises immediately. It is flammable and highly reactive with water (Worthy, 1985). MIC induced a mutagenic response in mouse lymphoma cells and chromosomal aberrations Correspondence: G.P. Meshram, Govt. of India Ministry of Defence, Research and Development Organisation, Defence Research and Development Establishment, Tansen Road, Gwalior-474 002 (India).
(CAs) and sister-chromatid exchanges (SCEs) in Chinese hamster ovary ceils (Caspary and Myhr, 1986; Mason et al., 1987). MIC is also reported to induce CAs, SCEs and micronuclei in bone marrow cells, SCEs in lung cells and peripheral blood lymphocytes of mice (Tice et al., 1987; Kligerman et al. 1987). From our preliminary studies, a weak mutagenic response of MIC was reported in Salmonella, with slight modification of preincubation test conditions (Meshram and Rao, 1986). However, recently Mason et al. (1987) reported a negative response in Salmonella, with standard preincubation protocol (37 ° C for 20 rain). MIC is a highly volatile chemical and it readily reacts with water at 37°C. For this reason, it is felt necessary to modify the test conditions of the
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
124 liquid-preincubation procedure to get an optimum mutagenic response of MIC in the Ames Salmonella mutagenicity test. Numerous modifications of the Ames test procedure have been reported to increase its sensitivity for selected compounds (Yahagi et al., 1975; Mitchell, 1978; Sugimura and Nagao, 1980; Rosenkranz et al., 1980). Matsushima et al. (1980) demonstrated the comparable results at 3 7 ° C for 20 min and 30 ° C for 30 min as test conditions in the liquid-preincubation procedure. For the assay with MIC we further decreased the temperature and increased the time of preincubation. In this paper, we report the results and data on the mutagenic response of MIC in the modified test conditions of the Ames Salmonella/microsome liquid-preincubation procedure. Materials and methods
Chemicals MIC (CAS No. 624-83-9) was synthesised in the chemistry division of this laboratory (Raghuveeran and Kaushik, 1985). Its purity was confirmed to be greater than 99% by gas chromatography. Cold dimethyl sulphoxide was added to the ampule containing 20 mg of MIC and different concentrations were prepared immediately, before its addition to the preincubation mixture. Dimethyl sulphoxide (DMSO), NADP, glucose 6-phosphate, 2-aminofluorene (2-AF), methyl methanesulphonate (MMS) and methylglyoxal were obtained from Sigma (St. Louis, MO, U.S.A.); 4-nitroquinoline N-oxide (NQNO) and 2,4,7-trinitro-9-fluorenone (TNF) from Fluka AG (Switzerland) and Aroclor 1254 from Analab (New Haven, CN, U.S.A.).
Gas-chromatographic analysis MIC is a highly volatile and reactive chemical. In the presence of excess of water, it reacts to form N, N'-dimethylurea. For the mutagenicity assay, although the initial concentrations of MIC are prepared in DMSO, finally it is added to the preincubation mixture, where MIC may react with excess water during the different test conditions of preincubation. For this reason, the stability of MIC in water was determined by gas chromatography, to find out the approximate per-
centage of MIC remaining unhydrolysed in water and to correlate its availability in the preincubation mixture under different test conditions. For this study Perkin-Elmer Gas Chromatograph model 3920, equipped with flame ionisation detector was used. The conditions of GC were as follows: injection port and detector maintained at 120°C, column at 105°C, nitrogen carrier gas flow at 20 ml/min. 4% MIC solution was prepared in premaintained distilled water at 1 0 ° C and 3 7 ° C and further incubated separately at respective temperatures. A 2-/~1 sample was injected into the column for GC analysis immediately after preparation of the solution and after different time intervals. Peak heights were measured and percentages of MIC remaining unhydrolysed in water were calculated by comparing with the peak height of a known volume of pure MIC. Stability data at 25 ° C were provided by Dr. R.C. Malhotra. Data on the stability of MIC in water at 3 incubation conditions are shown in Table 1.
Bacterial strains 5 histidine-deficient ( H i s - ) tester strains, TA97a, TA98, TA100, TA102 and TA104 of Salmonella typhimurium were kindly provided by Prof. B.N. Ames, Univ. of California, Berkeley, CA, U.S.A. 2 of these strains, TA97a and TA98, are used for the detection of frameshift mutagens and the remaining 3 for the detection of base pair substitution mutagens. Prior to use in the assay, TABLE 1 STABILITY OF MIC IN WATER AT 3 DIFFERENT INCUBATION TEMPERATURESAS ANALYSED BY GAS CHROMATOGRAPHY Time (rain)
Percentageof MIC detected in water at 37oc 25oc a 10oc
0 2 15 30 45 60 70
73 55 <2 -
ND 70 50 23 ND <1 -
a Unpublished data from Dr. R.C. Malhotra. ND, not determined.
100 97 75 45 13 5 <1
125
each strain was checked for the presence of strain-specific marker as described by Maron and Ames (1983). Bacterial cultures were inoculated from frozen permanents to 10 ml Oxoid nutrient broth No. 2 and incubated at 37°C for 16 h to obtain approximately 1 × 10 9 cells/ml.
Preparation of $9 Male Wistar rats weighing 150-200 g were pretreated with Aroclor 1254 and the $9 fraction and $9 mix were prepared following the procedure of Maron and Ames (1983). The protein concentration of the $9 fraction was 24 mg/ml as determined by the procedure of Lowry et al. (1951).
Mutagenicity assay The liquid-preincubation procedure described by Maron and Ames (1983) was used with slight
modification of the incubation test conditions. For the preparation of the preincubation mixture, the following ingredients were added in order to 15 mm × 125 mm sterile screw-capped glass culture tubes arranged in an ice bath, 0.5 ml either $9 mix (5% $9 fraction) or 0.02 M phosphate buffer (pH 7.4), 0.1 ml bacterial culture and 0.1 ml test chemical in DMSO. The preincubation mixture was incubated at 10 °C for 60 min. For comparative mutagenic activity of MIC, in TA100 and TA104 strains, 3 sets of preincubation mixture were prepared and incubated at 37 °C for 20 min, 20°C for 40 min and 10°C for 60 min respectively. Following incubation, 2 ml of molten top agar containing biotin and a trace amount of histidine was added to the preincubation mixture and poured on to minimal glucose agar plates. Plates were incubated at 37 °C and his- revertant
TABLE 2 M U T A G E N I C ACTIVITY OF M E T H Y L I S O C Y A N A T E IN Salmonella typhimurium W I T H M O D I F I E D TEST C O N D I T I O N S O F T H E L I Q U I D - P R E I N C U B A T I O N P R O C E D U R E (10 o C for 60 min) Test
Conc.
chemical
(#g/plate)
DMSO MIC
PC b PC b
$9
His ÷ revertants/plate (mean of 6 plates-+ S.E.) TA97a
TA98
TA100
TA102
-
+ -
171+ 160_+
6.0 5.1
36+ 35_+
1.6 1.8
186_+ 9.2 181_+ 4.5
500
+ -
185_+ 79+
8.4 2.5
34_+ 20-+
1.1 0.8
201_+ 7.4 (1.1) 114-+ 5.6 (0.6)
400
+ -
204_+ 162_+
5.8 7.5
41_+ 33_+
1.1 2.1
300
+ -
240_+ 165_+
4.1 4.8
35_+ 31_+
200
+ -
186_+ 156_+
3.6 6.1
100
+ -
177_+ 156_+
50
+ -
10
+ + -
TA104
219_+ 200+
6.6 4.5
635_+ 17 527-+ 10
216_+ 152_+
5.8 2.4
639_+ 20 ( 1 . 0 ) 395_+ 9.0(0.7)
218_+ 4.1 (1.2) 348_+ 6.1 (1.9)
199_+ 187_+
3.4 5.8
782_+ 16 953_+ 22
(1.2) (1.8)
1.3 1.6
288_+ 5.5 (1.5) 378_+ 7.8 (2.1)
241_+ 212_+
7.4 4.9
843_+ 15 1014_+ 22
(1.3) (1.9)
39_+ 34_+
1.7 1.3
310_+ 8.1 (1.7) 365+ 7.4 (2.0)
208_+ 211_+
8.7 6.0
9 4 7 + 22 1154_+ 30
(1.5) (2.2)
5.8 6.0
36_+ 37-+
1.7 1.4
347_+ 8.5 (1.9) 394-+ 7.6 (2.2)
239_+ 197+
6.7 5.5
1096_+ 22 1291-t- 26
(1.7) (2.4)
167-+ 168_+
3.1 5.7
35-+ 36_+
1.0 0.9
258-+ 6.1 (1.4) 304_+ 7.2 (1.7)
205+ 184_+
4.7 3.5
866_+ 17 1003_+ 12
(1.4) (1.9)
191_+ 152_+
4.9 2.7
33_+ 36+
1.3 2.2
216_+ 4.7 (1.2) 268_+ 6.4 (1.5)
221+ 197+
5.0 7.9
696-+ 13 905-+ 16
(1.1) (1.7)
ND 4077+145
(7.7)
ND 2424+101
3828_+ 84 4178-+100
2292-+85 3343_+91
(12) (18)
a
ND 3115-+117
a
a Revertant ratio (experimental/control). b Mean of 4 plates. N D , not determined; PC, positive control, without $9, TA97a and TA98, T N F - 0.25 #g; TA100, N Q N O - 0.5 /.tg; TA102 and TA104, MMS - 2.5/tl; with $9, TA98 and TA100, 2-AF - 20 /tg.
126 100
150C
~DO-"Q\
IO0 X\
A
40(3
\
b
) \
80
C \
\ \\
\\
©
_u 0_
120(
80
©
300
6O
El 90C
6O
0_
e
E El
c_ m
40 u~
2OO
> 60(
40
30(
20
©
t~ ]:
% 10C
\~
160 2bO 300 460
2O
T
5C)0"~
0
160 2bo 360 460 56d;%00
Dose (pg/Plate) 50C
Dose (#g/Plate) 100
~) \
1500 :OQ\
100
'o \
D
B 80
40C
120(
% \ \
~
80
©
o
30C
\\
60
EEl ©
900
6O
600
40 ~
i:1_
20C
40
10C
20
% I
©
"6
0
0 100 200 300 400 500 1000 Dose (#g/Plate)
% -
T
30(
0
\\~'\k
20
1()0 2C)03()0 460 5007000 ' 0 Dose (#g/Plate)
Fig. 1. Dose-response curve for MIC at 10 °C for 60-rain test conditions in TA100: (A) with $9; (B) without $9; TA104: (C) with $9; (D) without $9. O . . . . . . O, survival; O O, revertants/plate. colonies were scored after 48 h. MIC was assayed in triplicate at each dose. Mean numbers of revertant colonies of 2 separate assays + S.E. are listed in Table 2. A separate experiment was carried out for the dose-response mutagenic and toxic effects of MIC in TA100 and TA104 strains at 10 ° C for 60-min test conditions. Microcolonies (histidine auxotrophs) in the background lawn were examined in all the test plates and control (solvent) plates for the determination of toxicity of the test chemical.
Percentage of survivals was calculated as described by McGregor et al. (1984). Revertants were scored from the same plates for the determination of dose-response mutagenic effects of the test chemical. Values are the mean of 3 plates shown in Fig. 1. Results of the comparative mutagenic response of MIC in TA100 and TA104 strains at 3 test conditions are shown in Table 3. Concurrent solvent and positive controls were performed under similar test conditions along with each assay. Positive mutagenic response was con-
127 TABLE 3 COMPARATIVE M U T A G E N I C ACTIVITY OF METHYL ISOCYANATE IN TA100 A N D TA104 STRAINS AT 3 D I F F E R ENT PREINCUBATION TEST C O N D I T I O N S Test chemical
Conc. (tLg/plate)
$9
His + revertants/plate (mean of 3 plates_+ S.E.) TA100
TA104
37 ° C for 20 min DMSO
MIC
PC PC
20 o C for 40 rain
10 o C for 60 min
37 o C for 20 min
20 ° C for 40 min
10 o C for 60 min
-
+ -
157+ 146+
5.0 7.3
168+ 11.4 150_+ 6.3
164+ 8.6 152_+ 11
546__+ 15 475_+ 11
533+ 20 519_+ 27
542+ 14 503_+ 10
500
+ -
97_+ 94_+
7.2 5.9
131_+ 118_+
3.1 2.6
133_+ 125_+
5.0 7.6
371_+ 395_+
476_+ 16 507_+ 11
521_+ 21 495_+ 10
300
+ -
169_+ 174_+
6.7 2.7
199_+ 10 28l_+ 8.7
285_+ 355_+
3.6 5.0
533_+ 16 592_+ 11
832_+ 27 883-+ 14
934_+ 29 1019-+ 21
100
+ -
193_+ 209_+
6.5 5.3
267-+ 5.2 306_+ 12
308_+ 12 438_+ 7.8
626_+ 20 651+ 20
942_+ 41 1064_+ 26
1030_+ 22 1285+ 14
50
+ -
160_+ 160_+
8.3 4.9
233_+ 216_+
9.6 7.5
279-+ 13 323_+ 13
590+ 11 515_+ 12
842-+ 25 926_+ 19
951+ 36 1079_+ 45
10
+ -
146_+ 3.7 140-+ 10
155_+ 145-+
5.3 4.2
232_+ 12 249+- 9.0
533_+ 21 476_+ 19
576_+ 23 525__+ 24
601_+ 16 532-+ 27
2830_+ 71 6264_+132
3158+- 64 5775_+101
2855_+ 90 6161_+109
+ -
2087-+ 69 3336_+223
2077-+121 3303_+243
1886_+ 69 3273_+216
9.3 7.4
PC, positive control, without $9, TA100, N Q N O - 0.5 ~g; TA104, methylglyoxal - 50/~g; with $9, TA100 and TA104, 2-AF - 20 ~g.
sidered when the mean number of his + revertants obtained at any dose level was at least twice that of the solvent control. Results MIC induced mutagenic response in two base pair substitution strains TA100 and TA104 in the presence and absence of $9 at 1 0 ° C for 60-min test conditions. However, TA97a, TA98 and TA102 strains were unable to detect mutagenic activity of MIC both in the presence and absence of $9 (Table 2). At 1 0 ° C for the 60-min test condition, a reproducible dose-dependent increase in the number of revertants was observed in TA100 and TA104 strains with MIC at concentrations of 10-100 /zg/plate. With further increase in dose from 100 /~g/plate, a decrease in the number of revertants and percentage of survival was observed (Fig. 1). Incorporation of rat liver $9 mixture, resulted in a slight decrease in mutagenic and toxic response of MIC.
Results of the comparative mutagenic activity of MIC in TA100 and TA104 strains at 3 different test conditions of preincubation show that MIC induces mutagenic response at 1 0 ° C for 60-min and 20 ° C for 40-min test conditions. Maximum mutagenic response was observed at 1 0 ° C for 60-min test conditions. However, the response was negative at 37 ° C for 60-min test conditions, both in the presence and absence of $9. Discussion The results reveal that MIC induces mutagenic response in the modified test conditions of the Ames Salmonella microsome liquid-preincubation procedure. A reproducible dose-dependent increase in the number of revertants was observed in TA100 and TA104 strains. MIC is a highly reactive chemical and in the presence of excess water, it reacts to form N, N'dimethylurea. Methylamine and N-methylcarbamic acid are the intermediates in the hydrolysis of
128 M I C to N , N ' - d i m e t h y l u r e a . In vitro studies by Caspary and M y h r (1986) indicate that methylamine and N-methylcarbamic acid are weakly mutagenic and N , N ' - d i m e t h y l u r e a is not mutagenic in the mouse l y m p h o m a cell forward mutation assay. In the present study, when M I C is added to the preincubation mixture, it m a y react with excess water and m a y form more or less the same reaction products during preincubation in all 3 test conditions. However, the mutagenic activity of the reaction products of M I C observed by Caspary and M y h r (1986) cannot be readily attributed to the mutagenic activity of M I C observed in Salmonella in the modified test conditions of preincubation. Stability studies of M I C in water at 3 7 ° C , 2 5 ° C and 1 0 ° C , as determined by gas chromatography, show that a higher percentage of M I C remains unhydrolysed in water at 1 0 ° C c o m p a r e d to that at 2 5 ° C and 3 7 ° C . Stability data of M I C in water reveal that a higher percentage of M I C remains available for exposure to bacterial cells in the preincubation mixture at 1 0 ° C for 60-min test conditions, which could be the most likely reason for the higher mutagenic response observed at the same test conditions in comparison to that at 20 ° C for 40-min test conditions. The negative response of M I C was observed in Salmonella at 37 ° C for 20-min test conditions. Similar results were also reported recently by Mason et al. (1987) at the same test conditions. The m i n i m u m a m o u n t of M I C required to induce mutation in Salmonella could not be available at 37 ° C for 20-min test conditions due to loss of the chemical by evaporation and hydrolysis. However, the toxicity of M I C at higher concentration observed in Salmonella at all 3 test conditions might be due to M I C or its reaction products. These findings clearly indicate that the mutagenic response observed in the modified test conditions of the Ames S a l m o n e l l a / m i c r o s o m e liquid-preincubation procedure might be due to M I C itself or its u n k n o w n metabolites and not due to its main reaction products. MIC, being a carbamoylating agent, is electrophilic and can interact with D N A and proteins ( M a s o n et al., 1987), which m a y account for the mutagenic activity observed in the present study
and in various cytogenetic studies (Caspary and Myhr, 1986; Kligerman et al., 1987; M a s o n et al., 1987).
Acknowledgements We thank Dr. P.K. R a m a c h a n d r a n , Director, Defence Research and Development Establishment, Gwalior for active encouragement as well as Drs. R.C. Malhotra and M.V.S. S u r y a n a r a y a n a for G C analysis. The authors are also grateful to Dr. M.P. Kaushik for the synthesis and supply of pure methyl isocyanate for this study.
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., 31,347-364. Anon (1984) India's chemical tragedy: Death toll at Bhopal still rising, Chem. Eng. News, 62 (50), 6-8. Caspary, W.J., and B. Myhr (1986) Mutagenicity of methylisocyanate and its reaction products to culture mammalian cells, Mutation Res., 174, 285-293. Kligerman, A.D., J.A. Campbell, G.L. Erexson, J.W. Allen and M.D. Shelby (1987) Sister chromatid exchange analysis in lung and peripheral blood lymphocytes of mice exposed to methyl isocyanate by inhalation, Environ. Mutagen., 9, 29-36. Lowry, D.H., N.J. Rosebrough, A.L. Fan and R.J. Randall (1951) Protein measurement with folin phenol reagent, J. Biol. Chem., 193, 265-275. Maron, D.M., and B.N. Ames (1983) Revised method for Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mason, J.M., E. Zeiger, S. Hayworth, J. Ivett and R. Valencia (1987) Genotoxicity studies on methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells, Environ. Mutagen., 9, 19-28. Matsushima, T., T. Sugimura, M. Nagao, T. Yahagi, A. Shirai and M. Sawamura (1980) Factors modulating in microbial tests, in: K.H. Norpoth and R.C. Garner (Eds.), Short-Term Test System for Detecting Carcinogens, Springer, Berlin, pp. 273-285. McGregor, D., R.D. Prentice, M. McConville, Y.J. Lee and W.J. Caspary (1984) Reduced mutant yield at high doses in the Salmonella/activation assay: The cause is not always toxicity, Environ. Mutagen., 6, 545-557. Meshram, G.P., and K.M. Rao (1986) Preliminary studies on the mutagenic effects of methylisocyanate in the Ames test, llth Annual Conference of EMSI on Environmental Mutagens and Carcinogens, Madras (India), p. 10 (Abstr.). Mitchell, I. deG. (1978) Microbial assay for mutagenicity: A mofidied liquid culture method compared to the agar plate system for precision and sensitivity, Mutation Res., 54, 1-16.
129 Raghuveeran, C.D., and M.P. Kaushik (1985) Reversed-phase high-performance liquid chromatography of methylisocyanate, J. Chromatogr., 346, 446-449. Rosenkranz, H.S., G. Karpinsky and E.C. McCoy (1980) Microbial assay: Evaluation and application to the elucidation of the etiology of colon cancer, in: K. Norpoth and R.C. Garner (Eds.), Short-Term Test System for Detecting Carcinogens, Springer, Berlin, pp. 19-57. Sugimura, T., and M. Nagao (1980) Modification of mutagenic activity, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Principles and Methods for Their Detection, Plenum, New York, pp. 41-60.
Tice, R.R., C.A. Luke and M.D. Shelby (1987) Methyl isocyanate: An evaluation of in vivo cytogenetic activity, Environ. Mutagen., 9, 37-58. Worthy, W. (1985) Methylisocyanate: The chemistry of a hazard, Chem. Eng. News, 63(6), 27-33. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96.
123
MTR 01244
Mutagenicity of methyl isocyanate in the modified test conditions of Ames Salmonella/microsome liquid-preincubation procedure Ghansham P. Meshram and Kola M. Rao Division of Microbiology, Defence Research and Development Establishment, Tansen Road, Gwalior-474 002 (India) (Received 20 February 1987) (Revision received 11 August 1987) (Accepted 25 August 1987)
Keywords: Methyl isocyanate; Modified test conditions; Ames Salmonella/microsome liquid-preincubation procedure.
Summary Methyl isocyanate (MIC) was tested for mutagenicity using the Ames Salmonella/microsome liquidpreincubation procedure with slight modification of test conditions. In the modification the preincubation mixture was incubated at 10°C for 60 min. MIC was assayed both in the presence and absence of Aroclor-1254-induced $9, using 5 tester strains of Salmonella typhimurium, TA97a, TA98, TA100, TA102 and TA104. MIC induced mutagenic response in two base-pair substitution strains, TA100 and TA104, in the presence and absence of $9. However, mutagenic response in the presence of $9 was low as compared to that in the absence of $9. In the comparative mutagenic activity at 3 different preincubation test conditions (37 °C for 20 rain, 20 °C for 40 min and 10 °C for 60 min), optimum mutagenic response was observed at 10 °C for the 60-min test condition. However, no mutagenic response was observed at 37 °C for the 20-rain test condition.
Methyl isocyanate (MIC) is an industrial chemical used as a basic ingredient in the production of various pesticides such as aldicarb, carbaryl, carbofuran and methomyl (Anon, 1984). MIC is highly irritating and volatile. It has low boiling point (39.1 ° C) and high vapour pressure (348 mm Hg at 20 o C) and consequently, it vaporises immediately. It is flammable and highly reactive with water (Worthy, 1985). MIC induced a mutagenic response in mouse lymphoma cells and chromosomal aberrations Correspondence: G.P. Meshram, Govt. of India Ministry of Defence, Research and Development Organisation, Defence Research and Development Establishment, Tansen Road, Gwalior-474 002 (India).
(CAs) and sister-chromatid exchanges (SCEs) in Chinese hamster ovary ceils (Caspary and Myhr, 1986; Mason et al., 1987). MIC is also reported to induce CAs, SCEs and micronuclei in bone marrow cells, SCEs in lung cells and peripheral blood lymphocytes of mice (Tice et al., 1987; Kligerman et al. 1987). From our preliminary studies, a weak mutagenic response of MIC was reported in Salmonella, with slight modification of preincubation test conditions (Meshram and Rao, 1986). However, recently Mason et al. (1987) reported a negative response in Salmonella, with standard preincubation protocol (37 ° C for 20 rain). MIC is a highly volatile chemical and it readily reacts with water at 37°C. For this reason, it is felt necessary to modify the test conditions of the
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
124 liquid-preincubation procedure to get an optimum mutagenic response of MIC in the Ames Salmonella mutagenicity test. Numerous modifications of the Ames test procedure have been reported to increase its sensitivity for selected compounds (Yahagi et al., 1975; Mitchell, 1978; Sugimura and Nagao, 1980; Rosenkranz et al., 1980). Matsushima et al. (1980) demonstrated the comparable results at 3 7 ° C for 20 min and 30 ° C for 30 min as test conditions in the liquid-preincubation procedure. For the assay with MIC we further decreased the temperature and increased the time of preincubation. In this paper, we report the results and data on the mutagenic response of MIC in the modified test conditions of the Ames Salmonella/microsome liquid-preincubation procedure. Materials and methods
Chemicals MIC (CAS No. 624-83-9) was synthesised in the chemistry division of this laboratory (Raghuveeran and Kaushik, 1985). Its purity was confirmed to be greater than 99% by gas chromatography. Cold dimethyl sulphoxide was added to the ampule containing 20 mg of MIC and different concentrations were prepared immediately, before its addition to the preincubation mixture. Dimethyl sulphoxide (DMSO), NADP, glucose 6-phosphate, 2-aminofluorene (2-AF), methyl methanesulphonate (MMS) and methylglyoxal were obtained from Sigma (St. Louis, MO, U.S.A.); 4-nitroquinoline N-oxide (NQNO) and 2,4,7-trinitro-9-fluorenone (TNF) from Fluka AG (Switzerland) and Aroclor 1254 from Analab (New Haven, CN, U.S.A.).
Gas-chromatographic analysis MIC is a highly volatile and reactive chemical. In the presence of excess of water, it reacts to form N, N'-dimethylurea. For the mutagenicity assay, although the initial concentrations of MIC are prepared in DMSO, finally it is added to the preincubation mixture, where MIC may react with excess water during the different test conditions of preincubation. For this reason, the stability of MIC in water was determined by gas chromatography, to find out the approximate per-
centage of MIC remaining unhydrolysed in water and to correlate its availability in the preincubation mixture under different test conditions. For this study Perkin-Elmer Gas Chromatograph model 3920, equipped with flame ionisation detector was used. The conditions of GC were as follows: injection port and detector maintained at 120°C, column at 105°C, nitrogen carrier gas flow at 20 ml/min. 4% MIC solution was prepared in premaintained distilled water at 1 0 ° C and 3 7 ° C and further incubated separately at respective temperatures. A 2-/~1 sample was injected into the column for GC analysis immediately after preparation of the solution and after different time intervals. Peak heights were measured and percentages of MIC remaining unhydrolysed in water were calculated by comparing with the peak height of a known volume of pure MIC. Stability data at 25 ° C were provided by Dr. R.C. Malhotra. Data on the stability of MIC in water at 3 incubation conditions are shown in Table 1.
Bacterial strains 5 histidine-deficient ( H i s - ) tester strains, TA97a, TA98, TA100, TA102 and TA104 of Salmonella typhimurium were kindly provided by Prof. B.N. Ames, Univ. of California, Berkeley, CA, U.S.A. 2 of these strains, TA97a and TA98, are used for the detection of frameshift mutagens and the remaining 3 for the detection of base pair substitution mutagens. Prior to use in the assay, TABLE 1 STABILITY OF MIC IN WATER AT 3 DIFFERENT INCUBATION TEMPERATURESAS ANALYSED BY GAS CHROMATOGRAPHY Time (rain)
Percentageof MIC detected in water at 37oc 25oc a 10oc
0 2 15 30 45 60 70
73 55 <2 -
ND 70 50 23 ND <1 -
a Unpublished data from Dr. R.C. Malhotra. ND, not determined.
100 97 75 45 13 5 <1
125
each strain was checked for the presence of strain-specific marker as described by Maron and Ames (1983). Bacterial cultures were inoculated from frozen permanents to 10 ml Oxoid nutrient broth No. 2 and incubated at 37°C for 16 h to obtain approximately 1 × 10 9 cells/ml.
Preparation of $9 Male Wistar rats weighing 150-200 g were pretreated with Aroclor 1254 and the $9 fraction and $9 mix were prepared following the procedure of Maron and Ames (1983). The protein concentration of the $9 fraction was 24 mg/ml as determined by the procedure of Lowry et al. (1951).
Mutagenicity assay The liquid-preincubation procedure described by Maron and Ames (1983) was used with slight
modification of the incubation test conditions. For the preparation of the preincubation mixture, the following ingredients were added in order to 15 mm × 125 mm sterile screw-capped glass culture tubes arranged in an ice bath, 0.5 ml either $9 mix (5% $9 fraction) or 0.02 M phosphate buffer (pH 7.4), 0.1 ml bacterial culture and 0.1 ml test chemical in DMSO. The preincubation mixture was incubated at 10 °C for 60 min. For comparative mutagenic activity of MIC, in TA100 and TA104 strains, 3 sets of preincubation mixture were prepared and incubated at 37 °C for 20 min, 20°C for 40 min and 10°C for 60 min respectively. Following incubation, 2 ml of molten top agar containing biotin and a trace amount of histidine was added to the preincubation mixture and poured on to minimal glucose agar plates. Plates were incubated at 37 °C and his- revertant
TABLE 2 M U T A G E N I C ACTIVITY OF M E T H Y L I S O C Y A N A T E IN Salmonella typhimurium W I T H M O D I F I E D TEST C O N D I T I O N S O F T H E L I Q U I D - P R E I N C U B A T I O N P R O C E D U R E (10 o C for 60 min) Test
Conc.
chemical
(#g/plate)
DMSO MIC
PC b PC b
$9
His ÷ revertants/plate (mean of 6 plates-+ S.E.) TA97a
TA98
TA100
TA102
-
+ -
171+ 160_+
6.0 5.1
36+ 35_+
1.6 1.8
186_+ 9.2 181_+ 4.5
500
+ -
185_+ 79+
8.4 2.5
34_+ 20-+
1.1 0.8
201_+ 7.4 (1.1) 114-+ 5.6 (0.6)
400
+ -
204_+ 162_+
5.8 7.5
41_+ 33_+
1.1 2.1
300
+ -
240_+ 165_+
4.1 4.8
35_+ 31_+
200
+ -
186_+ 156_+
3.6 6.1
100
+ -
177_+ 156_+
50
+ -
10
+ + -
TA104
219_+ 200+
6.6 4.5
635_+ 17 527-+ 10
216_+ 152_+
5.8 2.4
639_+ 20 ( 1 . 0 ) 395_+ 9.0(0.7)
218_+ 4.1 (1.2) 348_+ 6.1 (1.9)
199_+ 187_+
3.4 5.8
782_+ 16 953_+ 22
(1.2) (1.8)
1.3 1.6
288_+ 5.5 (1.5) 378_+ 7.8 (2.1)
241_+ 212_+
7.4 4.9
843_+ 15 1014_+ 22
(1.3) (1.9)
39_+ 34_+
1.7 1.3
310_+ 8.1 (1.7) 365+ 7.4 (2.0)
208_+ 211_+
8.7 6.0
9 4 7 + 22 1154_+ 30
(1.5) (2.2)
5.8 6.0
36_+ 37-+
1.7 1.4
347_+ 8.5 (1.9) 394-+ 7.6 (2.2)
239_+ 197+
6.7 5.5
1096_+ 22 1291-t- 26
(1.7) (2.4)
167-+ 168_+
3.1 5.7
35-+ 36_+
1.0 0.9
258-+ 6.1 (1.4) 304_+ 7.2 (1.7)
205+ 184_+
4.7 3.5
866_+ 17 1003_+ 12
(1.4) (1.9)
191_+ 152_+
4.9 2.7
33_+ 36+
1.3 2.2
216_+ 4.7 (1.2) 268_+ 6.4 (1.5)
221+ 197+
5.0 7.9
696-+ 13 905-+ 16
(1.1) (1.7)
ND 4077+145
(7.7)
ND 2424+101
3828_+ 84 4178-+100
2292-+85 3343_+91
(12) (18)
a
ND 3115-+117
a
a Revertant ratio (experimental/control). b Mean of 4 plates. N D , not determined; PC, positive control, without $9, TA97a and TA98, T N F - 0.25 #g; TA100, N Q N O - 0.5 /.tg; TA102 and TA104, MMS - 2.5/tl; with $9, TA98 and TA100, 2-AF - 20 /tg.
126 100
150C
~DO-"Q\
IO0 X\
A
40(3
\
b
) \
80
C \
\ \\
\\
©
_u 0_
120(
80
©
300
6O
El 90C
6O
0_
e
E El
c_ m
40 u~
2OO
> 60(
40
30(
20
©
t~ ]:
% 10C
\~
160 2bO 300 460
2O
T
5C)0"~
0
160 2bo 360 460 56d;%00
Dose (pg/Plate) 50C
Dose (#g/Plate) 100
~) \
1500 :OQ\
100
'o \
D
B 80
40C
120(
% \ \
~
80
©
o
30C
\\
60
EEl ©
900
6O
600
40 ~
i:1_
20C
40
10C
20
% I
©
"6
0
0 100 200 300 400 500 1000 Dose (#g/Plate)
% -
T
30(
0
\\~'\k
20
1()0 2C)03()0 460 5007000 ' 0 Dose (#g/Plate)
Fig. 1. Dose-response curve for MIC at 10 °C for 60-rain test conditions in TA100: (A) with $9; (B) without $9; TA104: (C) with $9; (D) without $9. O . . . . . . O, survival; O O, revertants/plate. colonies were scored after 48 h. MIC was assayed in triplicate at each dose. Mean numbers of revertant colonies of 2 separate assays + S.E. are listed in Table 2. A separate experiment was carried out for the dose-response mutagenic and toxic effects of MIC in TA100 and TA104 strains at 10 ° C for 60-min test conditions. Microcolonies (histidine auxotrophs) in the background lawn were examined in all the test plates and control (solvent) plates for the determination of toxicity of the test chemical.
Percentage of survivals was calculated as described by McGregor et al. (1984). Revertants were scored from the same plates for the determination of dose-response mutagenic effects of the test chemical. Values are the mean of 3 plates shown in Fig. 1. Results of the comparative mutagenic response of MIC in TA100 and TA104 strains at 3 test conditions are shown in Table 3. Concurrent solvent and positive controls were performed under similar test conditions along with each assay. Positive mutagenic response was con-
127 TABLE 3 COMPARATIVE M U T A G E N I C ACTIVITY OF METHYL ISOCYANATE IN TA100 A N D TA104 STRAINS AT 3 D I F F E R ENT PREINCUBATION TEST C O N D I T I O N S Test chemical
Conc. (tLg/plate)
$9
His + revertants/plate (mean of 3 plates_+ S.E.) TA100
TA104
37 ° C for 20 min DMSO
MIC
PC PC
20 o C for 40 rain
10 o C for 60 min
37 o C for 20 min
20 ° C for 40 min
10 o C for 60 min
-
+ -
157+ 146+
5.0 7.3
168+ 11.4 150_+ 6.3
164+ 8.6 152_+ 11
546__+ 15 475_+ 11
533+ 20 519_+ 27
542+ 14 503_+ 10
500
+ -
97_+ 94_+
7.2 5.9
131_+ 118_+
3.1 2.6
133_+ 125_+
5.0 7.6
371_+ 395_+
476_+ 16 507_+ 11
521_+ 21 495_+ 10
300
+ -
169_+ 174_+
6.7 2.7
199_+ 10 28l_+ 8.7
285_+ 355_+
3.6 5.0
533_+ 16 592_+ 11
832_+ 27 883-+ 14
934_+ 29 1019-+ 21
100
+ -
193_+ 209_+
6.5 5.3
267-+ 5.2 306_+ 12
308_+ 12 438_+ 7.8
626_+ 20 651+ 20
942_+ 41 1064_+ 26
1030_+ 22 1285+ 14
50
+ -
160_+ 160_+
8.3 4.9
233_+ 216_+
9.6 7.5
279-+ 13 323_+ 13
590+ 11 515_+ 12
842-+ 25 926_+ 19
951+ 36 1079_+ 45
10
+ -
146_+ 3.7 140-+ 10
155_+ 145-+
5.3 4.2
232_+ 12 249+- 9.0
533_+ 21 476_+ 19
576_+ 23 525__+ 24
601_+ 16 532-+ 27
2830_+ 71 6264_+132
3158+- 64 5775_+101
2855_+ 90 6161_+109
+ -
2087-+ 69 3336_+223
2077-+121 3303_+243
1886_+ 69 3273_+216
9.3 7.4
PC, positive control, without $9, TA100, N Q N O - 0.5 ~g; TA104, methylglyoxal - 50/~g; with $9, TA100 and TA104, 2-AF - 20 ~g.
sidered when the mean number of his + revertants obtained at any dose level was at least twice that of the solvent control. Results MIC induced mutagenic response in two base pair substitution strains TA100 and TA104 in the presence and absence of $9 at 1 0 ° C for 60-min test conditions. However, TA97a, TA98 and TA102 strains were unable to detect mutagenic activity of MIC both in the presence and absence of $9 (Table 2). At 1 0 ° C for the 60-min test condition, a reproducible dose-dependent increase in the number of revertants was observed in TA100 and TA104 strains with MIC at concentrations of 10-100 /zg/plate. With further increase in dose from 100 /~g/plate, a decrease in the number of revertants and percentage of survival was observed (Fig. 1). Incorporation of rat liver $9 mixture, resulted in a slight decrease in mutagenic and toxic response of MIC.
Results of the comparative mutagenic activity of MIC in TA100 and TA104 strains at 3 different test conditions of preincubation show that MIC induces mutagenic response at 1 0 ° C for 60-min and 20 ° C for 40-min test conditions. Maximum mutagenic response was observed at 1 0 ° C for 60-min test conditions. However, the response was negative at 37 ° C for 60-min test conditions, both in the presence and absence of $9. Discussion The results reveal that MIC induces mutagenic response in the modified test conditions of the Ames Salmonella microsome liquid-preincubation procedure. A reproducible dose-dependent increase in the number of revertants was observed in TA100 and TA104 strains. MIC is a highly reactive chemical and in the presence of excess water, it reacts to form N, N'dimethylurea. Methylamine and N-methylcarbamic acid are the intermediates in the hydrolysis of
128 M I C to N , N ' - d i m e t h y l u r e a . In vitro studies by Caspary and M y h r (1986) indicate that methylamine and N-methylcarbamic acid are weakly mutagenic and N , N ' - d i m e t h y l u r e a is not mutagenic in the mouse l y m p h o m a cell forward mutation assay. In the present study, when M I C is added to the preincubation mixture, it m a y react with excess water and m a y form more or less the same reaction products during preincubation in all 3 test conditions. However, the mutagenic activity of the reaction products of M I C observed by Caspary and M y h r (1986) cannot be readily attributed to the mutagenic activity of M I C observed in Salmonella in the modified test conditions of preincubation. Stability studies of M I C in water at 3 7 ° C , 2 5 ° C and 1 0 ° C , as determined by gas chromatography, show that a higher percentage of M I C remains unhydrolysed in water at 1 0 ° C c o m p a r e d to that at 2 5 ° C and 3 7 ° C . Stability data of M I C in water reveal that a higher percentage of M I C remains available for exposure to bacterial cells in the preincubation mixture at 1 0 ° C for 60-min test conditions, which could be the most likely reason for the higher mutagenic response observed at the same test conditions in comparison to that at 20 ° C for 40-min test conditions. The negative response of M I C was observed in Salmonella at 37 ° C for 20-min test conditions. Similar results were also reported recently by Mason et al. (1987) at the same test conditions. The m i n i m u m a m o u n t of M I C required to induce mutation in Salmonella could not be available at 37 ° C for 20-min test conditions due to loss of the chemical by evaporation and hydrolysis. However, the toxicity of M I C at higher concentration observed in Salmonella at all 3 test conditions might be due to M I C or its reaction products. These findings clearly indicate that the mutagenic response observed in the modified test conditions of the Ames S a l m o n e l l a / m i c r o s o m e liquid-preincubation procedure might be due to M I C itself or its u n k n o w n metabolites and not due to its main reaction products. MIC, being a carbamoylating agent, is electrophilic and can interact with D N A and proteins ( M a s o n et al., 1987), which m a y account for the mutagenic activity observed in the present study
and in various cytogenetic studies (Caspary and Myhr, 1986; Kligerman et al., 1987; M a s o n et al., 1987).
Acknowledgements We thank Dr. P.K. R a m a c h a n d r a n , Director, Defence Research and Development Establishment, Gwalior for active encouragement as well as Drs. R.C. Malhotra and M.V.S. S u r y a n a r a y a n a for G C analysis. The authors are also grateful to Dr. M.P. Kaushik for the synthesis and supply of pure methyl isocyanate for this study.
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Tice, R.R., C.A. Luke and M.D. Shelby (1987) Methyl isocyanate: An evaluation of in vivo cytogenetic activity, Environ. Mutagen., 9, 37-58. Worthy, W. (1985) Methylisocyanate: The chemistry of a hazard, Chem. Eng. News, 63(6), 27-33. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96.