- Email: [email protected]
The activity of 4CMB, 4HMB and BC in Saccharomyces cerevisiae JD1
Mutation Research, 100 (1982) 163-168 Elsevier Biomedical Press
163
THE ACTIVITY OF 4CMB, 4HMB AND BC IN Saccharomyces cerevisiae JD1
PHILIP WILCOX and JAMES M. PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP, West Glamorgan (Great Britain)
(Received 14 April 1981) (Accepted 14 May 1981)
Summary 3 structurally related compounds, 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB), and benzyl chloride (BC) were assayed for their ability to induce mitotic gene conversion in stationary phase cultures o f the yeast, Saccharomyces cerevisiae JD1. This strain allows gene conversion to be scored at 2 independent loci, trp 5 and his 4. The results reported in this paper indicate that both 4CMB and BC are genetically active in yeast, producing dose-related increases in mitotic gene conversion at both the loci tested; 4HMB showed no such activity. At high survival levels 4CMB and BC showed comparable activity. However, as toxicity increased BC showed much more potent convertogenic activity, whereas with 4CMB a reduction in induced gene conversion was observed. The presence of a microsomal activation system derived from the livers of Aroclor-induced male rats did not significantly affect the activity of any of the compounds.
3 compounds, BC, 4HMB and 4CMB were examined for their ability to induce mitotic gene conversion in stationary phase cultures of the yeast Saccharomyces cerevisiae strain JD1. In this strain which is heteroallelic at loci tryptophan-5 and histidine-4, induced conversion can be monitored at 2 sites. The genotype of JD1 is given below; its use in detecting chemical and physical mutagens has been described in detail elsewhere (Davies et al., 1975). JD1
a ade2-1 c~ +
ser 7 +
his 8 +
his 4C his 4ABC
trp5-U9 trp5-U6
In our preliminary assays with 4CMB, a number of different protocols were investigated, including the use of exponential or stationary phase cells and exposure in either buffer or broth. In buffer, the exponential phase cells were found to be very 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
164
sensitive to the lethal effects of 4CMB and no increases in convertants per plate were observed. An increase in conversion frequency was apparent when calculated as convertants per surviving cell. However, stationary phase cells were more resistant to the lethal effects of 4CMB and up to 3-fold increases in the number of prototrophic colonies per plate were obtained. The exposure of growing yeast cells in nutrient broth to 4CMB gave inconsistent results. This was probably due to varying sensitivity to 4CMB throughout the yeast cell cycle and is being investigated further. An experiment was also carried out to establish the optimal length of exposure. Stationary phase cells in buffer were exposed to 3 different concentrations of 4CMB and incubated at 28 °C on an orbital shaker. Samples were removed every 2 h for a total of 18 h, and plated onto YC to determine survival and onto YM + his to score conversion at the trp 5 locus. The results o f this experiment clearly demonstrated that toxicity and induced gene conversion were still rising after 18 h with all 3 concentrations of 4CMB examined. It was, therefore, decided that treatment of stationary phase cells in buffer for 18 h was the most suitable method of exposure for studying the genetic activity of 4CMB in yeast cells. 4HMB and BC were also tested using several protocols. 4HMB gave negative results in all experiments and BC showed its greatest activity when cells were treated in buffer. The results reported in the present paper are those obtained when stationary phase cells in buffer were treated with 4CMB, 4HMB and BC for 18 h in the presence and absence of $9 mix. Materials and methods
Compounds 4CMB, 4HMB and BC were supplied by Dr. J. Ashby (I.C.I.). The compounds were dissolved in dimethyl sulphoxide (DMSO) and diluted in the same solvent to give the required concentrations. The concentrations used in this study are given below. These were selected on a basis of solubility, toxicity and maximum induction o f the genetic end-point. 4CMB: 125, 62.5, 31.3, 15.6 and 7.8/~g/ml; 4HMB" 250, 125, 62.5, 31.3 and 15.6 #g/ml; BC: 500, 250, 125, 62.5 and 31.3 #g/ml; Media Viability was scored on yeast complete (YC) agar plates. Conversion was scored on yeast minimal (YM) plates supplemented with histidine or tryptophan at 20 #g/ml. The buffer used was S0rensen phosphate buffer pH 7.0. Treatment conditions 4-day-old colonies grown on solid YC medium were suspended in pH 7.0 phosphate buffer at approximately 3 x 10 7 cells/ml. This suspension was divided into 1.9-ml aliquots in 30-ml universal bottles (Sterilin) and 0.1 ml of test compound solution was added. For the $9 experiments, 0.1 ml o f test compound was added to 1.6 ml of yeast suspension and 0.3 ml of $9 mix. The tubes were incubated for 18 h
165 at 28 °C on an orbital incubator and then washed twice in saline 2 ml saline. Cells were plated directly on to YM + his and conversion (5 plates per treatment), and diluted and plated on viability (3 plates per treatment). The YC plates were counted YM plates after 5 - 6 days incubation at 28 °C.
and resuspended in YM + trp to score YC plates to score after 2 - 3 days and
$9 mix The $9 mix used contained 1 ml of Aroclor-induced $9 fraction, 9 ml p H 7.0 buffer and 30 mg N A D P H . In all assays using $9, a known positive control compound, cyclophosphamide (250 #g/ml), was included to check that the $9 was capable of metabolic activation. On all occasions this gave the expected results, i.e. a 4 - 5 - f o l d increase in conversion frequency. Results The results obtained with 4CMB, 4HMB and BC are given in Fig. 1. In the absence of $9 mix, 4CMB induced dose-related increases in the frequency of mitotic conversion at both the trp 5 and his 4 locus, amounting to approximately 5 times and 4 times the solvent control value, respectively. Survival was reduced to 65% at the highest dose tested, i.e. 125 #g/ml. The presence of $9 mix did not significantly affect the activity of 4CMB. A decrease in conversion frequency was observed with 4CMB at 125/~g/ml in the presence and the absence of $9. This decrease was also obtained in repeat assays and may indicate that at high concentrations 4CMB is killing the cells by a non-genetic mechanism. This effect is being investigated further and will be the subject of a future paper. 4HMB was found to be non-toxic and non-convertogenic at concentrations up to 250/~g/ml both in the presence and the absence of $9 mix. BC produced clear doserelated increases in gene conversion at both loci. At concentrations which gave low toxicity the increases were comparable to those obtained with 4CMB. However, at concentrations of 250 and 500/~g/ml survival was reduced to 48% and 9% respectively, and large increases in gene conversion were observed. As also shown with 4CMB, the presence of $9 did not appear to significantly affect the activity of BC. In bacterial mutagenicity assays, the presence of $9 considerably reduced the mutagenic activity of 4CMB (Ashby et al., 1981). However, in our experiments, with stationary phase cells no such reduction was observed. To investigate this apparent difference, a further experiment was carried out in which 4CMB was preincubated with $9 mix. Selected concentrations of 4CMB were incubated at 37 °C with normal $9 mix, heat-inactivated $9 mix, and cofactors alone. After 3 h,-stationary phase yeast cells suspended in p H 7.0 buffer were added to each tube and incubation continued at 28 °C for 18 h on an orbital shaker. Cells were then washed twice and plated to score conversion at the trp-5 locus and cell survival. The results of this experiment are given in Fig. 2. The curves obtained following pre-incubation with normal $9 mix, heat-inactivated $9 mix and cofactors alone were similar. In
166
+S9
-$9
o
7:8 1~.6 31'.3 62.s 125 2~o 560 -S9
j 500u.I
/"
0400
0
o
500400-
/
300-
//
200-
300-
Z8 15',6 3113 6;~.5 1;~5 250 500
100O-+ql , o 7.e 80
-
-
15~ 31136i5 125 25o 5oo
$9
/
u
o
6O
z < t-- 40-
g u 4-
0~ 80,
7% 15'.63£3 6:;.5 ~2s 2so 5oo
+$ 9
/o
/ 60-
o'
40
i 20-
20
-tO "~'~'~-~t
0
-
;
-
"
78 15'.6 31~3 6::;5 12S 2~)0 500 CONCENTRATION ( p g / m l )
O~
7:8 15'.6 311302.5 1;)5 250 500 CONCENTRATION ( ~ g / m l )
Fig. 1. Survival and induction o f mitotic gene conversion in stationary phase cells o f S. cerevisiae JDI in the presence and absence o f $9. A , 4 C M B - $ 9 ; I I , 4 H M B - $ 9 ; . , BC - $ 9 ; A , 4 C M B + $ 9 ; t~, 4 H M B + $ 9 ; o, BC + $ 9 .
the presence of normal $9 mix the number of trp ÷ convertants was slightly reduced at low concentrations and slightly increased at high concentrations, but as these effects were also observed with the heat-inactivated $9 preparation, they probably
167
PRE-INCUBATION
WITH
~
NORMAL
/
7060-
U
o
<>
50-
"-- 40-
0 300
u)
+
LO
o
59
2010 ~
0
-II 0
0
7.e, 15'.6 31t3 62,5 125
PR E - I N C U B A T I O N
~1 0
WITH
24
zh ~g.6 3~t3 62%
INACTIVE
~6
sg
70u~ j 60.J
u
~>
~3 50" 40" > Z 30O 0 20-
to O O1-
~_
,
~ 10O
O' "~dt 0
z~ 15:6 31:3 6;~.5 ~s
PRE-INCUBATION
WITH
~'8 ~5'.6 3;.3 d.s 4s
COFACTORS
70 ¸ J w
60.
'0 5 0 0 I -~40~
>
~ 30
S
+ 20"
0
~o-q
o o[[/~ 0
¢8 ~6'.631'.3 6~t.5 ~5 Conch (pg/rnl)
---q/
7~8 15'.6 31'.3 62.5 125 Concn (~g / r n l )
and induction of mitotic gene conversion in stationary phase cells pre-incubation with normal $9, heat-inactivated $9 and cofactors a l o n e .
F i g . 2. S u r v i v a l following
O-
o f S. cerevisiae JDI
reflect minor differences in treatment conditions, such as a reduction in the effective concentration o f 4 C M B due to binding to $9 proteins, and the presence o f tryptophan in the $9 allowing s o m e residual cell growth on the minimal plates. It, therefore, appears that even after pre-incubation, S9 has little effect on 4CMB-induced mitotic gene conversion.
168
Conclusions From the results o f these experiments, it is concluded that both 4CMB and BC induce mitotic gene conversion in stationary phase cells of the yeast S. cerevisiae strain JD1; in contrast 4HMB showed no such activity in this assay. At high viability levels 4CMB and BC showed comparable activity. However, as toxicity increased BC showed much more potent convertogenic activity, whereas with 4CMB a reduction in gene conversion was observed. The presence of $9 did not significantly affect the activity of any of the compounds.
Acknowledgements One of us (PW) was in receipt of an SRC studentship. The work was supported by funds from the EEC Environmental Programme.
References Ashby, J., R.W. Trueman, J. Styles, M.G. Penman and D. Paton (1981) 4-Chloromethylbiphenyl (4CMB): a novel mutagen and potential carcinogen, Carcinogenesis, 2, 33-38. Davies, P.J., W.E. Evans and J.M. Parry (1975) Mitotic recombination induced by chemical and physical agents in the yeast Saccharornyces cerevisiae, Mutation Res., 29, 301-314.
163
THE ACTIVITY OF 4CMB, 4HMB AND BC IN Saccharomyces cerevisiae JD1
PHILIP WILCOX and JAMES M. PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP, West Glamorgan (Great Britain)
(Received 14 April 1981) (Accepted 14 May 1981)
Summary 3 structurally related compounds, 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB), and benzyl chloride (BC) were assayed for their ability to induce mitotic gene conversion in stationary phase cultures o f the yeast, Saccharomyces cerevisiae JD1. This strain allows gene conversion to be scored at 2 independent loci, trp 5 and his 4. The results reported in this paper indicate that both 4CMB and BC are genetically active in yeast, producing dose-related increases in mitotic gene conversion at both the loci tested; 4HMB showed no such activity. At high survival levels 4CMB and BC showed comparable activity. However, as toxicity increased BC showed much more potent convertogenic activity, whereas with 4CMB a reduction in induced gene conversion was observed. The presence of a microsomal activation system derived from the livers of Aroclor-induced male rats did not significantly affect the activity of any of the compounds.
3 compounds, BC, 4HMB and 4CMB were examined for their ability to induce mitotic gene conversion in stationary phase cultures of the yeast Saccharomyces cerevisiae strain JD1. In this strain which is heteroallelic at loci tryptophan-5 and histidine-4, induced conversion can be monitored at 2 sites. The genotype of JD1 is given below; its use in detecting chemical and physical mutagens has been described in detail elsewhere (Davies et al., 1975). JD1
a ade2-1 c~ +
ser 7 +
his 8 +
his 4C his 4ABC
trp5-U9 trp5-U6
In our preliminary assays with 4CMB, a number of different protocols were investigated, including the use of exponential or stationary phase cells and exposure in either buffer or broth. In buffer, the exponential phase cells were found to be very 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
164
sensitive to the lethal effects of 4CMB and no increases in convertants per plate were observed. An increase in conversion frequency was apparent when calculated as convertants per surviving cell. However, stationary phase cells were more resistant to the lethal effects of 4CMB and up to 3-fold increases in the number of prototrophic colonies per plate were obtained. The exposure of growing yeast cells in nutrient broth to 4CMB gave inconsistent results. This was probably due to varying sensitivity to 4CMB throughout the yeast cell cycle and is being investigated further. An experiment was also carried out to establish the optimal length of exposure. Stationary phase cells in buffer were exposed to 3 different concentrations of 4CMB and incubated at 28 °C on an orbital shaker. Samples were removed every 2 h for a total of 18 h, and plated onto YC to determine survival and onto YM + his to score conversion at the trp 5 locus. The results o f this experiment clearly demonstrated that toxicity and induced gene conversion were still rising after 18 h with all 3 concentrations of 4CMB examined. It was, therefore, decided that treatment of stationary phase cells in buffer for 18 h was the most suitable method of exposure for studying the genetic activity of 4CMB in yeast cells. 4HMB and BC were also tested using several protocols. 4HMB gave negative results in all experiments and BC showed its greatest activity when cells were treated in buffer. The results reported in the present paper are those obtained when stationary phase cells in buffer were treated with 4CMB, 4HMB and BC for 18 h in the presence and absence of $9 mix. Materials and methods
Compounds 4CMB, 4HMB and BC were supplied by Dr. J. Ashby (I.C.I.). The compounds were dissolved in dimethyl sulphoxide (DMSO) and diluted in the same solvent to give the required concentrations. The concentrations used in this study are given below. These were selected on a basis of solubility, toxicity and maximum induction o f the genetic end-point. 4CMB: 125, 62.5, 31.3, 15.6 and 7.8/~g/ml; 4HMB" 250, 125, 62.5, 31.3 and 15.6 #g/ml; BC: 500, 250, 125, 62.5 and 31.3 #g/ml; Media Viability was scored on yeast complete (YC) agar plates. Conversion was scored on yeast minimal (YM) plates supplemented with histidine or tryptophan at 20 #g/ml. The buffer used was S0rensen phosphate buffer pH 7.0. Treatment conditions 4-day-old colonies grown on solid YC medium were suspended in pH 7.0 phosphate buffer at approximately 3 x 10 7 cells/ml. This suspension was divided into 1.9-ml aliquots in 30-ml universal bottles (Sterilin) and 0.1 ml of test compound solution was added. For the $9 experiments, 0.1 ml o f test compound was added to 1.6 ml of yeast suspension and 0.3 ml of $9 mix. The tubes were incubated for 18 h
165 at 28 °C on an orbital incubator and then washed twice in saline 2 ml saline. Cells were plated directly on to YM + his and conversion (5 plates per treatment), and diluted and plated on viability (3 plates per treatment). The YC plates were counted YM plates after 5 - 6 days incubation at 28 °C.
and resuspended in YM + trp to score YC plates to score after 2 - 3 days and
$9 mix The $9 mix used contained 1 ml of Aroclor-induced $9 fraction, 9 ml p H 7.0 buffer and 30 mg N A D P H . In all assays using $9, a known positive control compound, cyclophosphamide (250 #g/ml), was included to check that the $9 was capable of metabolic activation. On all occasions this gave the expected results, i.e. a 4 - 5 - f o l d increase in conversion frequency. Results The results obtained with 4CMB, 4HMB and BC are given in Fig. 1. In the absence of $9 mix, 4CMB induced dose-related increases in the frequency of mitotic conversion at both the trp 5 and his 4 locus, amounting to approximately 5 times and 4 times the solvent control value, respectively. Survival was reduced to 65% at the highest dose tested, i.e. 125 #g/ml. The presence of $9 mix did not significantly affect the activity of 4CMB. A decrease in conversion frequency was observed with 4CMB at 125/~g/ml in the presence and the absence of $9. This decrease was also obtained in repeat assays and may indicate that at high concentrations 4CMB is killing the cells by a non-genetic mechanism. This effect is being investigated further and will be the subject of a future paper. 4HMB was found to be non-toxic and non-convertogenic at concentrations up to 250/~g/ml both in the presence and the absence of $9 mix. BC produced clear doserelated increases in gene conversion at both loci. At concentrations which gave low toxicity the increases were comparable to those obtained with 4CMB. However, at concentrations of 250 and 500/~g/ml survival was reduced to 48% and 9% respectively, and large increases in gene conversion were observed. As also shown with 4CMB, the presence of $9 did not appear to significantly affect the activity of BC. In bacterial mutagenicity assays, the presence of $9 considerably reduced the mutagenic activity of 4CMB (Ashby et al., 1981). However, in our experiments, with stationary phase cells no such reduction was observed. To investigate this apparent difference, a further experiment was carried out in which 4CMB was preincubated with $9 mix. Selected concentrations of 4CMB were incubated at 37 °C with normal $9 mix, heat-inactivated $9 mix, and cofactors alone. After 3 h,-stationary phase yeast cells suspended in p H 7.0 buffer were added to each tube and incubation continued at 28 °C for 18 h on an orbital shaker. Cells were then washed twice and plated to score conversion at the trp-5 locus and cell survival. The results of this experiment are given in Fig. 2. The curves obtained following pre-incubation with normal $9 mix, heat-inactivated $9 mix and cofactors alone were similar. In
166
+S9
-$9
o
7:8 1~.6 31'.3 62.s 125 2~o 560 -S9
j 500u.I
/"
0400
0
o
500400-
/
300-
//
200-
300-
Z8 15',6 3113 6;~.5 1;~5 250 500
100O-+ql , o 7.e 80
-
-
15~ 31136i5 125 25o 5oo
$9
/
u
o
6O
z < t-- 40-
g u 4-
0~ 80,
7% 15'.63£3 6:;.5 ~2s 2so 5oo
+$ 9
/o
/ 60-
o'
40
i 20-
20
-tO "~'~'~-~t
0
-
;
-
"
78 15'.6 31~3 6::;5 12S 2~)0 500 CONCENTRATION ( p g / m l )
O~
7:8 15'.6 311302.5 1;)5 250 500 CONCENTRATION ( ~ g / m l )
Fig. 1. Survival and induction o f mitotic gene conversion in stationary phase cells o f S. cerevisiae JDI in the presence and absence o f $9. A , 4 C M B - $ 9 ; I I , 4 H M B - $ 9 ; . , BC - $ 9 ; A , 4 C M B + $ 9 ; t~, 4 H M B + $ 9 ; o, BC + $ 9 .
the presence of normal $9 mix the number of trp ÷ convertants was slightly reduced at low concentrations and slightly increased at high concentrations, but as these effects were also observed with the heat-inactivated $9 preparation, they probably
167
PRE-INCUBATION
WITH
~
NORMAL
/
7060-
U
o
<>
50-
"-- 40-
0 300
u)
+
LO
o
59
2010 ~
0
-II 0
0
7.e, 15'.6 31t3 62,5 125
PR E - I N C U B A T I O N
~1 0
WITH
24
zh ~g.6 3~t3 62%
INACTIVE
~6
sg
70u~ j 60.J
u
~>
~3 50" 40" > Z 30O 0 20-
to O O1-
~_
,
~ 10O
O' "~dt 0
z~ 15:6 31:3 6;~.5 ~s
PRE-INCUBATION
WITH
~'8 ~5'.6 3;.3 d.s 4s
COFACTORS
70 ¸ J w
60.
'0 5 0 0 I -~40~
>
~ 30
S
+ 20"
0
~o-q
o o[[/~ 0
¢8 ~6'.631'.3 6~t.5 ~5 Conch (pg/rnl)
---q/
7~8 15'.6 31'.3 62.5 125 Concn (~g / r n l )
and induction of mitotic gene conversion in stationary phase cells pre-incubation with normal $9, heat-inactivated $9 and cofactors a l o n e .
F i g . 2. S u r v i v a l following
O-
o f S. cerevisiae JDI
reflect minor differences in treatment conditions, such as a reduction in the effective concentration o f 4 C M B due to binding to $9 proteins, and the presence o f tryptophan in the $9 allowing s o m e residual cell growth on the minimal plates. It, therefore, appears that even after pre-incubation, S9 has little effect on 4CMB-induced mitotic gene conversion.
168
Conclusions From the results o f these experiments, it is concluded that both 4CMB and BC induce mitotic gene conversion in stationary phase cells of the yeast S. cerevisiae strain JD1; in contrast 4HMB showed no such activity in this assay. At high viability levels 4CMB and BC showed comparable activity. However, as toxicity increased BC showed much more potent convertogenic activity, whereas with 4CMB a reduction in gene conversion was observed. The presence of $9 did not significantly affect the activity of any of the compounds.
Acknowledgements One of us (PW) was in receipt of an SRC studentship. The work was supported by funds from the EEC Environmental Programme.
References Ashby, J., R.W. Trueman, J. Styles, M.G. Penman and D. Paton (1981) 4-Chloromethylbiphenyl (4CMB): a novel mutagen and potential carcinogen, Carcinogenesis, 2, 33-38. Davies, P.J., W.E. Evans and J.M. Parry (1975) Mitotic recombination induced by chemical and physical agents in the yeast Saccharornyces cerevisiae, Mutation Res., 29, 301-314.