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Genetic effects induced in Sacharomyces cerevisiae by cyclophosphamide in vitro without liver enzyme preparations
201
Mutation Research, 37 (1976) 201--212 © Elsevier/North-Holland Biomedical Press
GENETIC EFFECTS INDUCED IN SA CCHAR 034 YCES CERE VISIAE BY CYCLOPHOSPHAMIDE IN VITRO WITHOUT LIVER ENZYME PREPARATIONS
V.W. MAYER, C.J. HYBNER and D.J. BRUSICK 1 Genetic Toxicology Branch, Division of Toxicology, Food and Drug Administration, Department of Health, Education, and Welfare, Washington, D.C. 20204 and 1 Genetics Department, Litton Bionetics Inc., 5516 Nicholson Lane, Kensington, Maryland 20795 (U.S.A.) (Received January 15th, 1976) (Revision received June 10th, 1976) (Accepted June 23rd, 1976)
Summary Cyclophosphamide induced forward mutation in Saccharomyces cerevisiae strain $288C and mitotic recombination in strains D3 and D5 but not in strain D4. The yeast cells were treated with the c o m p o u n d in phosphate buffer without recourse to metabolic activation protocols. Elevation of the treatment temperature increased the genetic activity of cyclophosphamide. Respiration-deficient isolates of strains $288C and D3 were more sensitive than the respiratory competerit parent strains were for inducing forward mutation and mitotic recombination, respectively. Cyclophosphamide was incubated in phosphate buffer alone for increasing time intervals; strain D3 cells were added to aliquots for each time interval and incubated for an additional 30 min. The frequency of induced recombination increased as the time of c o m p o u n d incubation increased, showing that spontaneous degradation of cyclophosphamide to genetically active breakdown products was responsible for the genetic damage induced in the yeast cells.
Introduction The in vivo biological activity of cyclophosphamide (2-(bis(2-chloroethyl)amino) tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) is due to its metabolic conversion to active breakdown products by microsomal mixed-function oxidase enzymes [11,12,22,44]. Since unconverted cyclophosphamide is an inactive transport moiety, its breakdown is considered to be responsible for its mutagenic activity. Earlier studies demonstrated genetic activity with cyclo-
202 phosphamide in mice [9,45], Drosophila melanogaster [7,18,37], Chinese hamster bone marrow in vivo [21,39], murine and human cells in vivo [6,20] and in Vicia faba [31]. More recent studies have been designed specifically to take advantage of mammalian metabolism in the formation of mutagenic breakdown products from cyclophosphamide. Fahrig studied the ability of cyclophosphamide to induce mitotic gene conversion in strain D4 using mice [14] and rats [15] in exploratory studies with the host-mediated assay technique [19]. Gene conversion could be enhanced by blocking renal tubular excretion of the genetically active metabolites with probenecid [41] or by delaying cyclophosphamide breakdown using carbon tetrachloride or ethanol as hepatotoxins [8]. Similarly, various bacterial strains have been used to study the mutagenic activity of cyclophosphamide after conversion to its active form in a host-mediated assay [36,38]. Ellenberger and Mohn [13] found that cyclophosphamide was not active per se b u t was genetically active in mutation tests with bacteria only after biotransformation by rodent liver homogenates. Genetic activity of cyclophosphamide was discovered by combining yeast strain D4 with the urine of treated rats [26,40] ; however, no activity could be found if the yeast cells were treated in vitro. These studies led logically to testing for genetically active metabolites in b o d y fluids obtained from a human patient receiving cyclophosphamide therapy. Genetic activity was found in the urine [42,43] b u t not in ascitic fluid [43]. Thus there is a considerable b o d y of knowledge on in vivo genetic activity, all of which strongly suggests that cyclophosphamide is genetically active only after metabolic conversion by mammals. Cyclophosphamide is also known to be degraded in aqueous media either by heating the solution [4,16,17,31] or under physiological conditions [5]. The c o m p o u n d is also broken down in vitro by microsomal enzyme treatment as well as by several chemical oxidizing systems [1,2,44]. Interest in the spontaneous breakdown of cyclophosphamide prompted us to investigate this compound for possible genetic effects without resorting to metabolic activation protocols. The results are reported here. Materials and methods
Saccharomyces cerevisiae strains A haploid strain designated $288C (a,p÷, can s) was used to observe forward mutation from canavanine sensitivity to canavanine resistance as previously described [25]. Induced reciprocal mitotic recombination was observed in diploid strain D3 [52] and strain D5 [48]. The use of the ade2, his8 and CYH4 markers in the heterozygous strain D3 [30,53] and the heteroallelic ade2 markers in strain D5 [48,49] for detection of mitotic recombination was previously described. Induced mitotic gene conversion was observed using diploid strain D4 which is heteroallelic at the ade2 and trp5 loci [51]. Spontaneous petite mutants of strains $288C and D3 were isolated from complete medium plates by their appearance as small (petite) colonies. The petite p h e n o t y p e was confirmed by their inability to reduce 2,3,5-triphenyltetrazolium chloride [34] and by their inability to grow on a non-fermentable carbon source [33]. These strains were used to observe canavanine resistance, mutation induction and mitotic recombination induction in the appropriate strains with a petite cytoplasmic background.
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Media and chemicals All yeast strains were routinely cultivated on a semisynthetic complete medium described by Pittman et al. [35]. The same medium was used to detect the red-sectored colonies resulting from mitotic recombination in strains D3 and D5. Mitotic recombination in the petite isolate of strain D3 was detected on the semisynthetic complete medium containing 8% glucose [23]. Canavanine-resistant mutants were detected on minimal medium [24] containing Lcanavanine sulfate at 30 pg/ml [25], which selectively allows only canavanineresistant mutants to grow. Selective media consisting of minimal medium [24] supplemented with adenine sulfate (30 pg/ml) was used to identify strain D4 colonies with trp5 convertant alleles. A synthetic complete medium either lacking histidine or having, in addition, 1 pg/ml cycloheximide was used to test for homozygosity of the two peripheral markers to ade2, his8 and CYH4, respectively, in strain D3 as previously described [30]. Cyclophosphamide was obtained from Mead Johnson and Co., Evansville, Indiana, and solutions of appropriate concentration were prepared immediately before use. Cycloheximide and L-canavanine sulfate were obtained from Calbiochem, San Diego, California. Experimental design Treatment with cyclophosphamide All treatments of the various yeast strains were conducted using 10 mg/ml cyclophosphamide in 0.067 M phosphate buffer at pH 7.2. Yeast cells to be used in experiments were grown on semisynthetic complete medium for 72 h at 30°C to achieve a confluent lawn of growth. The cells were rinsed from the agar surface and washed twice in phosphate buffer, and the inoculum was standardized as previously described [28]. A different procedure was followed to prepare strain D4 for experiments and was detailed previously [49]. Approximately 5 X 107 strain D3 and D5 cells/ml and 5 X l 0 s cells/ml of strain $288C were suspended in phosphate buffer and cyclophosphamide was added at the designated concentration. Samples were treated for selected increments of time (0, 2, 4 and 6 h) and temperature (23 and 30°C) on a rotary shaker at 200 rpm. Forward mutation induction Strain $288C or its petite isolate was used. Samples from the treatment flasks were diluted and plated according to standard microbiological techniques. Low dilutions were plated onto minimal medium plus canavanine for the detection of canavanine-resistant mutants and higher dilutions were plated onto semisynthetic complete medium for determination of the surviving population. Colonies were scored after 5 days of incubation at 30°C [29] for the wild type $288C, whereas 6 days of incubation was required for the petite isolate. Gene conversion in strain D4 After treatment, samples were diluted and plated on appropriate medium. Low dilutions were plated on minimal medium plus adenine for the detection of prototropic convertants at the trp5 locus. High dilutions were plated onto
204 semisynthetic complete medium for enumeration of the surviving population. Colonies were enumerated after incubation at 30°C for 3 days.
Mitotic recombination in strain D5 After treatment, samples were diluted and plated onto semisynthetic complete medium. Appropriate dilutions were made so that a sufficient number of colonies could be screened for sectors and proper definition of the red and pink sectors could be differentiated [48,49]. Petri dishes from further dilutions were used to enumerate the surviving population. Plates were incubated for 2 days at 30°C and held at 4°C for an additional 2 days, which seemed to promote color development before scoring. Plates containing sectored colonies were scored using a dissecting microscope at 10× magnification [30]. Mitotic recombination in strain D3 Samples were diluted and plated on semisynthetic complete medium, incubated and scored for red-sectored colonies as previously described [28]. The induction of red-sectored colonies was also observed in the petite isolate of strain D3 using semisynthetic complete medium with 8% glucose. Petri plates were incubated at 30°C for 3 days before scoring. Further tests were performed on a representative sample of sectored colonies from the non-petite strain D3 to show that the his8 and CYH4 markers became homozygous simultaneously with the ade2 marker. Such tests give further evidence that mitotic crossingover is the mechanism responsible for the red-sectored colonies and not mutation or gene conversion. The genetic basis for this evidence along with the procedure has been documented previously [30,50,52,53]. Effect of incubating cyclophosphamide in buffer alone for increasing time intervals Although it is possible that cyclophosphamide was genetically active directly in certain strains of S. cerevisiae, it is more likely on the basis of available evidence that the c o m p o u n d was breaking down spontaneously in phosphate buffer, yielding genetically active breakdown products. An experiment was designed to distinguish between these two possibilities. Separate aliquots of cyclophosphamide (10 mg/ml) were incubated in phosphate buffer for increasing time intervals at 30°C before adding the yeast cells. At time 0, 0.5, 1, 2, 4 and 6 h, 5 X 107 cells/ml of strain D3 were added to each flask and all flasks were incubated for an additional 0.5 h. The spontaneous control value was measured in another sample in which yeast cells were incubated for 0.5 h in phosphate buffer alone. A sample from each flask was diluted and plated immediately after the 0.5 h treatment. The plates were incubated and scored as described above. It was anticipated that a constant frequency of induced sectored colonies would be observed among the samples if spontaneous breakdown of cyclophosphamide were not related to its mutagenic activity, since the yeast cells were exposed to the chemical for 0.5 h in each case. However, if cyclophosphamide is breaking down during incubation in buffer, then sequential samples should show increasing genetic activity in the yeast cells.
205 TABLE I E F F E C T OF 10 m g / m l C Y C L O P H O S P H A M I D E ON I N D U C T I O N O F F O R W A R D M U T A T I O N IN S. CEREVISIAE STRAIN $288C Treatment conditions
Treatment time i n t e r v a l (h)
Percent survival
Population screened
Can R mutants
Can R / 105 survivors
G r a n d e (p+) 3 0 ° C
0 2 4 6
100 117 121 100
2.3 2.7 2.8 2.3
X X × ×
108 108 108 108
44 59 155 212
18.7 21.9 55.4 92.2
Petite ( p - ) 2 3 ° C
0 2 4 6
100 84 71 65
3.1 2.6 2.2 2.0
X X X ×
108 108 108 108
41 79 146 369
13.2 30.4 66.4 184.5
Petite ( p - ) 3 0 ° C
0 2 4 6
100 88 68 36
2.5 2.2 1.7 0.9
× X X ×
108 108 108 108
38 283 1417 142
15.2 128.6 833.5 157.8
Results Cyclophosphamide caused an increase in the frequency of forward mutation in the haploid S. cerevisiae strain, as can be observed in Table I. Somewhat higher mutation frequencies and correspondingly reduced survival were observed in the petite strain (p-) than in the wild type (p+) strain. The data further indicate that cyclophosphamide shows greater mutagenicity at the higher treatment temperature in the petite strain. In contrast to the experiments for mutagenicity, cyclophosphamide caused no increase in the frequency of gene conversion in strain D4 (Table II). No differences were observed either between the untreated control samples and the cyclophosphamide-treated samples or between samples treated at room temperature (23 ° C) and at 30 ° C.
T A B L E II I N E F F E C T I V E N E S S O F 10 m g / m l C Y C L O P H O S P H A M I D E ON I N D U C T I O N O F G E N E C O N V E R S I O N IN S. C E R E V I S I A E S T R A I N D4 Treatment time i n t e r v a l (h)
Temperature
Percent survival
Population screened
Trp + convertants
Trp+/10 s survivors
6 (control) 2 4 6
23°C
100 82 91 77
5.6 4.6 5.1 4.3
X × × ×
107 107 107 107
530 442 532 726
0.95 0.96 1.04 1.69
6 (control) 2 4 6
30°C
100 90 90 84
7.7 6.9 6.9 6.5
× X X X
107 107 107 107
402 413 403 452
0.52 0.60 0.58 0.70
Percent survival
i00
86
79
70
Treatment time interval (h)
0
2
4
6
4.4 X 104
5.0 X 104
5.4 X 104
6.3 × 104
Population screened
133 (0.30%)
3 (0.00%) 73 (0.13%) 111 (0.22%)
Red and Pink
107 (0.24%)
8 (0.01%) 38 (0.07%) 110 (0.22%)
Red
162 (0.36%)
9 (0.01%) 42 (0.07%) 110 (0.25%)
Pink
149 (0.33%)
6 (0.01%) 96 (0.17%) 125 (0.25%)
Pink and White
E F F E C T OF 10 m g / m l C Y C L O P H O S P H A M I D E A T 3 0 ° C O N I N D U C T I O N O F M I T O T I C R E C O M B I N A T I O N IN S. C E R E V I S I A E
TABLE III
133 (0.30%)
17 (0.03%) 123 (0.22%) 140 (0.28%)
Red and White
STRAIN D5
683 (1.55%)
43 (O.O6%) 372 (0.68%) 594 (1.18%)
Total altered colonies
b0
207 TABLE IV E F F E C T O F 10 m g / m l C Y C L O P H O S P H A M I D E S. C E R E V I S I A E S T R A I N D 3
ON INDUCTION OF MITOTIC RECOMBINATION
Treatment conditions
Treatment time i n t e r v a l (h)
Percent survival
Population screened
R e d sectoted colonies
G r a n d e (p+) 23°C
0 2 4 6
100 109 103 100
3.4 3.7 3.5 3.4
X X X X
10 l0 l0 l0
s s s s
44 101 224 399
12.9 27.3 64.0 117.4
G r a n d e (p+) 30°C
0 2 4 6
100 91 85 96
4.6 4.2 3.9 4.4
X X X X
l0 l0 l0 l0
s s s s
86 386 732 1304
18.7 91.9 187.7 296.4
Petite ( p - ) 23°C
0 2 4 6
100 63 63 67
3.0 1.9 1.9 2.0
X X × X
l0 s 10 s 10 s l0 s
62 143 332 583
20.7 75.3 174.7 291.5
Petite ( p - ) 30°C
0 2 4 6
100 63 63 53
3.0 1.9 1.9 1.6
X X X X
105 10 s 105 10 s
62 502 1382 2500
20.7 264.2 727.4 1562.5
IN
Sectors/ l0 s survivors
In strain D5, cyclophosphamide induced increases in the frequency of all the various types (Table III). The red-pink sectors represent the class of confirmed reciprocal crossover events, whereas the other sector types arise from undetermined genetic origins b u t are most probably due to gene conversion or mutation [48]. Reciprocal mitotic crossing-over was also induced in S. cerevisiae strain D3 (Table IV). The increase in red-sectored colonies observed with the time of exposure of the yeast cells to the c o m p o u n d showed a higher frequency at the higher temperature (30°C) than at room temperature (23°C). As was found in
TABLE V E F F E C T O F I N C U B A T I N G C Y C L O P H O S P H A M I D E IN B U F F E R A T 3 0 ° C F O R V A R Y I N G L E N G T H S O F T I M E P R I O R T O A D D I N G S. C E R E V I S 1 A E S T R A I N D3 T O M O N I T O R I N D U C E D M I T O T I C RECOMBINATION S t r a i n D3 a d d e d at d e s i g n a t e d t i m e s a n d a s s a y e d a f t e r 0.5 h i n c u b a t i o n w i t h c y c l o p h o s p h a m i d e . Cyclophosphamide i n c u b a t i o n t i m e (h)
Population screened
Sectored colonies
Sectors/10 s survivors
0 (buffer only) 0 0.5 1 2 4 6
2.8 2.5 2.6 2.8 2.8 2.5 2.7
73 113 139 176 213 192 218
26.1 45.2 53.5 62.9 76.1 76.8 80.7
X X × X X × X
l0 s l0 s l0 s 105 10 s l0 s 10 S
208 90 [
80
70
? >
6O
~o 50
/
4O
30
0
015 1
1;
2.5
45
6J5
Total cyclophosphomide incubatron time (hours) Fig. 1. E f f e c t o f i n c u b a t i n g e y c l o p h o s p h a m i d e in b u f f e r at 3 0 ° C f o r increasing l e n g t h s of t i m e p r i o r to the a d d i t i o n of strain D3 cells for 0.5 h t o m o n i t o r i n d u c e d m i t o t i c r e c o m b i n a t i o n .
the forward mutation experiments using strain $288C, the petite (p-) m u t a n t isolate of strain D3 was more sensitive to the mutagenic activity of cyclophosphamide than was the parent (p*) strain. The red portion and the white portion of 53 sectored colonies induced by cyclophosphamide were isolated and tested for induced homozygosity of markers peripheral to the ade2 marker. In the homozygous red sectors 50 were also homozygous for histidine requirement, and in the white sectors the marker for cycloheximide sensitivity was homozygous in 51 of the isolates. Such results are expected if the red-sectored colonies are due to reciprocal mitotic crossing-over [30,53]. The few clones from red-sectored colonies which did not show the reciprocal homozygous phenotypes are most likely due to gene conversion or mutation at the ade2 locus [30,53]. The genetic activity of cyclophosphamide increased when the c o m p o u n d was incubated for increasing lengths of time in phosphate buffer without yeast cells (Table V). The increase in sector frequency can be more readily observed in Fig. 1. Sector frequency increased until approximately 2.5 h and then remained stable at that elevated level in the 4.5 and 6.5 h samples. Discussion
Cyclophosphamide induces genetic damage in several strains of S. cerevisiae without the requirement for mammalian metabolism to produce genetically active breakdown products. These findings are in contrast to most of the litera-
209 ture concerning the mutagenesis of this substance in a wide variety of test organisms, including bacteria [13,36,38] and yeast [8,14,15,41,42,43]. Tests for mutagenicity of cyclophosphamide using bacteria and yeast have been consistently negative unless mammalian metabolism was provided, either as a liver enzyme preparation [13] or in studies involving the intact animal [15,26,36, 40]. With one exception, a preliminary report from our laboratory [10], no strains of S. cerevisiae other than D4 have heretofore been thoroughly tested in vitro with cyclophosphamide. Our experience reported here confirms the fact that no genetic activity could be demonstrated for cyclophosphamide in strain D4 without some provision for mammalian metabolism [15,26,40,41--43]. The strain D4 used in the above investigations is respiratory-deficient (as is our own strain) and we considered the possibility that respiratory competence is necessary for in vitro cyclophosphamide activity. Accordingly we isolated petite clones from strains $288C and D3, anticipating that genetic tests in these isolates might be negative. Instead they showed a greater response than did the corresponding respiration-sufficient parent strains. It is worthy of note that plate tests without liver activation using yeast strain D4 (for gene conversion), strain $288C (for forward mutation) and strains $211 and S138 (both for reverse mutation) were all negative with cyclophosphamide [10]. Furthermore, plate tests using strain D4 with liver homogenate preparations were negative (D. Brusick, unpublished observations) in contrast to the numerous citations to positive results in comparable liquid suspension tests with strain D4. These results suggest that yeast plate tests, at least with some compounds, may be less sensitive to genetic damage than are comparable liquid suspension tests. Similar experience has been reported in bacterial plate tests with dimethyl- and diethylnitrosamine [-3]. On the other hand some compounds appear to be more sensitive in plate tests than in liquid suspension tests [3,32] {D. Brusick, unpublished observations). Strain $288C was used to measure forward mutation, while strains D3 and D5 measure with complete confidence only reciprocal mitotic crossing-over. Strain D4, in contrast, measures only gene conversion and from a superficial consideration of our data alone it would appear that without metabolic degradation cyclophosphamide can induce mutation and crossing-over but not gene conversion. On the other hand cyclophosphamide does indeed induce gene conversion in strain D4 when metabolic activation is included in the experimental method [15,26,40--43]. Furthermore, many of the sectored colonies observed in strains D3 and D5 that did not show the appropriate reciprocal products associated with mitotic crossingover were most probably due to gene conversion events. These studies suggest that cyclophosphamide does not preferentially induce one class of genetic event over another or that strain D4 is unique in some unknown respect. In our studies the respiration-deficient isolates of strains $288C and D3 were more sensitive to the genetic activity of cyclophosphamide than were the respiration-competent parent strains. Such sensitivity has been observed before [46, 47] and a possible reason for this sensitivity has been advanced [46]. This observation may deserve further attention for the development of strains with enhanced sensitivity to chemical mutagens. It should be noted, however, that the respiration-deficient phenotype may interfere with the expression of the
210
nuclear genetic effect under study. Such was the case in strain D3 in which it was necessary to use 8% instead of the usual 2% glucose [23] and to strictly follow the schedule for incubation of petri dishes and enumeration of sectored colonies. The red in the respiration-deficient isolate developed, then faded rapidly, but the color was quite stable in the respiration-competent strain D3. The canavanine-resistant phenotype was not affected by the respiration condition of the strain $288C cells. Experiments on the mutagenicity of cyclophosphamide were conducted at room temperature (23°C) and at 30°C. It was anticipated that elevating the treatment temperature would increase the mutagenicity either by accelerating the spontaneous degradation of cyclophosphamide or by enhancing the metabolism of the yeast cells to take up and distribute the c o m p o u n d more rapidly. Such increases were observed in experiments with strains $288C and D3; however, no differences were found in D4. A similar enhancement of genetic activity by elevation of the treatment temperature has been previously observed for chromosome aberrations in Vicia faba [31]. Consideration of the rather extensive literature describing the in vitro breakdc~,~n of cyclophosphamide [1,2,4,5,16,17,27,44] and consideration of our own mutagenicity data reported here leads us to believe that the compound is not active per se, but requires degradation, either spontaneously in aqueous solution or metabolically in the liver and perhaps other organs. An experiment was designed to observe more directly the possibility of spontaneous degradation of cyclophosphamide to form genetically active breakdown products. Aliquots of cyclophosphamide in buffer were incubated for increasing time intervals before strain D3 cells were added for an additional 0.5 h of incubation. If cyclophosphamide were active per se and not degraded to genetically active breakdown products, then the same frequency of sectored colonies would be expected in all treated samples because the yeast cells would all be exposed to the compound for the same length of time. If, however, the genetic activity were dependent on the accumulation over a period of time of active breakdown products, then later samples would show greater genetic activity than earlier samples. Our experiments demonstrated an increasing frequency of induced recombination from 0 to 2.5 h after which no further increase was apparent. Thus the in vitro genetic activity of cyclophosphamide for S. cerevisiae is dependent on spontaneously generated active breakdown products. It appears from early studies [4,5] and from more recent observations [16, 17] that the pathways of spontaneous degradation of cyclophosphamide differ from metabolic activation pathways and result in the formation of different end products in each case. The fact that some end products may be identical for both in vitro and in vivo processes is also noted [1]; however, it is possible that strain D4 is responsive only to end products formed by in vivo processes while the other yeast strains used can respond to end products formed in the spontaneous degradation in aqueous medium. These experiments raise questions about the role played by the spontaneously generated breakdown of cyclophosphamide in in vivo processes such as antineoplastic activity or genetic activity in mammals.
211
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212
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Mutation Research, 37 (1976) 201--212 © Elsevier/North-Holland Biomedical Press
GENETIC EFFECTS INDUCED IN SA CCHAR 034 YCES CERE VISIAE BY CYCLOPHOSPHAMIDE IN VITRO WITHOUT LIVER ENZYME PREPARATIONS
V.W. MAYER, C.J. HYBNER and D.J. BRUSICK 1 Genetic Toxicology Branch, Division of Toxicology, Food and Drug Administration, Department of Health, Education, and Welfare, Washington, D.C. 20204 and 1 Genetics Department, Litton Bionetics Inc., 5516 Nicholson Lane, Kensington, Maryland 20795 (U.S.A.) (Received January 15th, 1976) (Revision received June 10th, 1976) (Accepted June 23rd, 1976)
Summary Cyclophosphamide induced forward mutation in Saccharomyces cerevisiae strain $288C and mitotic recombination in strains D3 and D5 but not in strain D4. The yeast cells were treated with the c o m p o u n d in phosphate buffer without recourse to metabolic activation protocols. Elevation of the treatment temperature increased the genetic activity of cyclophosphamide. Respiration-deficient isolates of strains $288C and D3 were more sensitive than the respiratory competerit parent strains were for inducing forward mutation and mitotic recombination, respectively. Cyclophosphamide was incubated in phosphate buffer alone for increasing time intervals; strain D3 cells were added to aliquots for each time interval and incubated for an additional 30 min. The frequency of induced recombination increased as the time of c o m p o u n d incubation increased, showing that spontaneous degradation of cyclophosphamide to genetically active breakdown products was responsible for the genetic damage induced in the yeast cells.
Introduction The in vivo biological activity of cyclophosphamide (2-(bis(2-chloroethyl)amino) tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) is due to its metabolic conversion to active breakdown products by microsomal mixed-function oxidase enzymes [11,12,22,44]. Since unconverted cyclophosphamide is an inactive transport moiety, its breakdown is considered to be responsible for its mutagenic activity. Earlier studies demonstrated genetic activity with cyclo-
202 phosphamide in mice [9,45], Drosophila melanogaster [7,18,37], Chinese hamster bone marrow in vivo [21,39], murine and human cells in vivo [6,20] and in Vicia faba [31]. More recent studies have been designed specifically to take advantage of mammalian metabolism in the formation of mutagenic breakdown products from cyclophosphamide. Fahrig studied the ability of cyclophosphamide to induce mitotic gene conversion in strain D4 using mice [14] and rats [15] in exploratory studies with the host-mediated assay technique [19]. Gene conversion could be enhanced by blocking renal tubular excretion of the genetically active metabolites with probenecid [41] or by delaying cyclophosphamide breakdown using carbon tetrachloride or ethanol as hepatotoxins [8]. Similarly, various bacterial strains have been used to study the mutagenic activity of cyclophosphamide after conversion to its active form in a host-mediated assay [36,38]. Ellenberger and Mohn [13] found that cyclophosphamide was not active per se b u t was genetically active in mutation tests with bacteria only after biotransformation by rodent liver homogenates. Genetic activity of cyclophosphamide was discovered by combining yeast strain D4 with the urine of treated rats [26,40] ; however, no activity could be found if the yeast cells were treated in vitro. These studies led logically to testing for genetically active metabolites in b o d y fluids obtained from a human patient receiving cyclophosphamide therapy. Genetic activity was found in the urine [42,43] b u t not in ascitic fluid [43]. Thus there is a considerable b o d y of knowledge on in vivo genetic activity, all of which strongly suggests that cyclophosphamide is genetically active only after metabolic conversion by mammals. Cyclophosphamide is also known to be degraded in aqueous media either by heating the solution [4,16,17,31] or under physiological conditions [5]. The c o m p o u n d is also broken down in vitro by microsomal enzyme treatment as well as by several chemical oxidizing systems [1,2,44]. Interest in the spontaneous breakdown of cyclophosphamide prompted us to investigate this compound for possible genetic effects without resorting to metabolic activation protocols. The results are reported here. Materials and methods
Saccharomyces cerevisiae strains A haploid strain designated $288C (a,p÷, can s) was used to observe forward mutation from canavanine sensitivity to canavanine resistance as previously described [25]. Induced reciprocal mitotic recombination was observed in diploid strain D3 [52] and strain D5 [48]. The use of the ade2, his8 and CYH4 markers in the heterozygous strain D3 [30,53] and the heteroallelic ade2 markers in strain D5 [48,49] for detection of mitotic recombination was previously described. Induced mitotic gene conversion was observed using diploid strain D4 which is heteroallelic at the ade2 and trp5 loci [51]. Spontaneous petite mutants of strains $288C and D3 were isolated from complete medium plates by their appearance as small (petite) colonies. The petite p h e n o t y p e was confirmed by their inability to reduce 2,3,5-triphenyltetrazolium chloride [34] and by their inability to grow on a non-fermentable carbon source [33]. These strains were used to observe canavanine resistance, mutation induction and mitotic recombination induction in the appropriate strains with a petite cytoplasmic background.
203
Media and chemicals All yeast strains were routinely cultivated on a semisynthetic complete medium described by Pittman et al. [35]. The same medium was used to detect the red-sectored colonies resulting from mitotic recombination in strains D3 and D5. Mitotic recombination in the petite isolate of strain D3 was detected on the semisynthetic complete medium containing 8% glucose [23]. Canavanine-resistant mutants were detected on minimal medium [24] containing Lcanavanine sulfate at 30 pg/ml [25], which selectively allows only canavanineresistant mutants to grow. Selective media consisting of minimal medium [24] supplemented with adenine sulfate (30 pg/ml) was used to identify strain D4 colonies with trp5 convertant alleles. A synthetic complete medium either lacking histidine or having, in addition, 1 pg/ml cycloheximide was used to test for homozygosity of the two peripheral markers to ade2, his8 and CYH4, respectively, in strain D3 as previously described [30]. Cyclophosphamide was obtained from Mead Johnson and Co., Evansville, Indiana, and solutions of appropriate concentration were prepared immediately before use. Cycloheximide and L-canavanine sulfate were obtained from Calbiochem, San Diego, California. Experimental design Treatment with cyclophosphamide All treatments of the various yeast strains were conducted using 10 mg/ml cyclophosphamide in 0.067 M phosphate buffer at pH 7.2. Yeast cells to be used in experiments were grown on semisynthetic complete medium for 72 h at 30°C to achieve a confluent lawn of growth. The cells were rinsed from the agar surface and washed twice in phosphate buffer, and the inoculum was standardized as previously described [28]. A different procedure was followed to prepare strain D4 for experiments and was detailed previously [49]. Approximately 5 X 107 strain D3 and D5 cells/ml and 5 X l 0 s cells/ml of strain $288C were suspended in phosphate buffer and cyclophosphamide was added at the designated concentration. Samples were treated for selected increments of time (0, 2, 4 and 6 h) and temperature (23 and 30°C) on a rotary shaker at 200 rpm. Forward mutation induction Strain $288C or its petite isolate was used. Samples from the treatment flasks were diluted and plated according to standard microbiological techniques. Low dilutions were plated onto minimal medium plus canavanine for the detection of canavanine-resistant mutants and higher dilutions were plated onto semisynthetic complete medium for determination of the surviving population. Colonies were scored after 5 days of incubation at 30°C [29] for the wild type $288C, whereas 6 days of incubation was required for the petite isolate. Gene conversion in strain D4 After treatment, samples were diluted and plated on appropriate medium. Low dilutions were plated on minimal medium plus adenine for the detection of prototropic convertants at the trp5 locus. High dilutions were plated onto
204 semisynthetic complete medium for enumeration of the surviving population. Colonies were enumerated after incubation at 30°C for 3 days.
Mitotic recombination in strain D5 After treatment, samples were diluted and plated onto semisynthetic complete medium. Appropriate dilutions were made so that a sufficient number of colonies could be screened for sectors and proper definition of the red and pink sectors could be differentiated [48,49]. Petri dishes from further dilutions were used to enumerate the surviving population. Plates were incubated for 2 days at 30°C and held at 4°C for an additional 2 days, which seemed to promote color development before scoring. Plates containing sectored colonies were scored using a dissecting microscope at 10× magnification [30]. Mitotic recombination in strain D3 Samples were diluted and plated on semisynthetic complete medium, incubated and scored for red-sectored colonies as previously described [28]. The induction of red-sectored colonies was also observed in the petite isolate of strain D3 using semisynthetic complete medium with 8% glucose. Petri plates were incubated at 30°C for 3 days before scoring. Further tests were performed on a representative sample of sectored colonies from the non-petite strain D3 to show that the his8 and CYH4 markers became homozygous simultaneously with the ade2 marker. Such tests give further evidence that mitotic crossingover is the mechanism responsible for the red-sectored colonies and not mutation or gene conversion. The genetic basis for this evidence along with the procedure has been documented previously [30,50,52,53]. Effect of incubating cyclophosphamide in buffer alone for increasing time intervals Although it is possible that cyclophosphamide was genetically active directly in certain strains of S. cerevisiae, it is more likely on the basis of available evidence that the c o m p o u n d was breaking down spontaneously in phosphate buffer, yielding genetically active breakdown products. An experiment was designed to distinguish between these two possibilities. Separate aliquots of cyclophosphamide (10 mg/ml) were incubated in phosphate buffer for increasing time intervals at 30°C before adding the yeast cells. At time 0, 0.5, 1, 2, 4 and 6 h, 5 X 107 cells/ml of strain D3 were added to each flask and all flasks were incubated for an additional 0.5 h. The spontaneous control value was measured in another sample in which yeast cells were incubated for 0.5 h in phosphate buffer alone. A sample from each flask was diluted and plated immediately after the 0.5 h treatment. The plates were incubated and scored as described above. It was anticipated that a constant frequency of induced sectored colonies would be observed among the samples if spontaneous breakdown of cyclophosphamide were not related to its mutagenic activity, since the yeast cells were exposed to the chemical for 0.5 h in each case. However, if cyclophosphamide is breaking down during incubation in buffer, then sequential samples should show increasing genetic activity in the yeast cells.
205 TABLE I E F F E C T OF 10 m g / m l C Y C L O P H O S P H A M I D E ON I N D U C T I O N O F F O R W A R D M U T A T I O N IN S. CEREVISIAE STRAIN $288C Treatment conditions
Treatment time i n t e r v a l (h)
Percent survival
Population screened
Can R mutants
Can R / 105 survivors
G r a n d e (p+) 3 0 ° C
0 2 4 6
100 117 121 100
2.3 2.7 2.8 2.3
X X × ×
108 108 108 108
44 59 155 212
18.7 21.9 55.4 92.2
Petite ( p - ) 2 3 ° C
0 2 4 6
100 84 71 65
3.1 2.6 2.2 2.0
X X X ×
108 108 108 108
41 79 146 369
13.2 30.4 66.4 184.5
Petite ( p - ) 3 0 ° C
0 2 4 6
100 88 68 36
2.5 2.2 1.7 0.9
× X X ×
108 108 108 108
38 283 1417 142
15.2 128.6 833.5 157.8
Results Cyclophosphamide caused an increase in the frequency of forward mutation in the haploid S. cerevisiae strain, as can be observed in Table I. Somewhat higher mutation frequencies and correspondingly reduced survival were observed in the petite strain (p-) than in the wild type (p+) strain. The data further indicate that cyclophosphamide shows greater mutagenicity at the higher treatment temperature in the petite strain. In contrast to the experiments for mutagenicity, cyclophosphamide caused no increase in the frequency of gene conversion in strain D4 (Table II). No differences were observed either between the untreated control samples and the cyclophosphamide-treated samples or between samples treated at room temperature (23 ° C) and at 30 ° C.
T A B L E II I N E F F E C T I V E N E S S O F 10 m g / m l C Y C L O P H O S P H A M I D E ON I N D U C T I O N O F G E N E C O N V E R S I O N IN S. C E R E V I S I A E S T R A I N D4 Treatment time i n t e r v a l (h)
Temperature
Percent survival
Population screened
Trp + convertants
Trp+/10 s survivors
6 (control) 2 4 6
23°C
100 82 91 77
5.6 4.6 5.1 4.3
X × × ×
107 107 107 107
530 442 532 726
0.95 0.96 1.04 1.69
6 (control) 2 4 6
30°C
100 90 90 84
7.7 6.9 6.9 6.5
× X X X
107 107 107 107
402 413 403 452
0.52 0.60 0.58 0.70
Percent survival
i00
86
79
70
Treatment time interval (h)
0
2
4
6
4.4 X 104
5.0 X 104
5.4 X 104
6.3 × 104
Population screened
133 (0.30%)
3 (0.00%) 73 (0.13%) 111 (0.22%)
Red and Pink
107 (0.24%)
8 (0.01%) 38 (0.07%) 110 (0.22%)
Red
162 (0.36%)
9 (0.01%) 42 (0.07%) 110 (0.25%)
Pink
149 (0.33%)
6 (0.01%) 96 (0.17%) 125 (0.25%)
Pink and White
E F F E C T OF 10 m g / m l C Y C L O P H O S P H A M I D E A T 3 0 ° C O N I N D U C T I O N O F M I T O T I C R E C O M B I N A T I O N IN S. C E R E V I S I A E
TABLE III
133 (0.30%)
17 (0.03%) 123 (0.22%) 140 (0.28%)
Red and White
STRAIN D5
683 (1.55%)
43 (O.O6%) 372 (0.68%) 594 (1.18%)
Total altered colonies
b0
207 TABLE IV E F F E C T O F 10 m g / m l C Y C L O P H O S P H A M I D E S. C E R E V I S I A E S T R A I N D 3
ON INDUCTION OF MITOTIC RECOMBINATION
Treatment conditions
Treatment time i n t e r v a l (h)
Percent survival
Population screened
R e d sectoted colonies
G r a n d e (p+) 23°C
0 2 4 6
100 109 103 100
3.4 3.7 3.5 3.4
X X X X
10 l0 l0 l0
s s s s
44 101 224 399
12.9 27.3 64.0 117.4
G r a n d e (p+) 30°C
0 2 4 6
100 91 85 96
4.6 4.2 3.9 4.4
X X X X
l0 l0 l0 l0
s s s s
86 386 732 1304
18.7 91.9 187.7 296.4
Petite ( p - ) 23°C
0 2 4 6
100 63 63 67
3.0 1.9 1.9 2.0
X X × X
l0 s 10 s 10 s l0 s
62 143 332 583
20.7 75.3 174.7 291.5
Petite ( p - ) 30°C
0 2 4 6
100 63 63 53
3.0 1.9 1.9 1.6
X X X X
105 10 s 105 10 s
62 502 1382 2500
20.7 264.2 727.4 1562.5
IN
Sectors/ l0 s survivors
In strain D5, cyclophosphamide induced increases in the frequency of all the various types (Table III). The red-pink sectors represent the class of confirmed reciprocal crossover events, whereas the other sector types arise from undetermined genetic origins b u t are most probably due to gene conversion or mutation [48]. Reciprocal mitotic crossing-over was also induced in S. cerevisiae strain D3 (Table IV). The increase in red-sectored colonies observed with the time of exposure of the yeast cells to the c o m p o u n d showed a higher frequency at the higher temperature (30°C) than at room temperature (23°C). As was found in
TABLE V E F F E C T O F I N C U B A T I N G C Y C L O P H O S P H A M I D E IN B U F F E R A T 3 0 ° C F O R V A R Y I N G L E N G T H S O F T I M E P R I O R T O A D D I N G S. C E R E V I S 1 A E S T R A I N D3 T O M O N I T O R I N D U C E D M I T O T I C RECOMBINATION S t r a i n D3 a d d e d at d e s i g n a t e d t i m e s a n d a s s a y e d a f t e r 0.5 h i n c u b a t i o n w i t h c y c l o p h o s p h a m i d e . Cyclophosphamide i n c u b a t i o n t i m e (h)
Population screened
Sectored colonies
Sectors/10 s survivors
0 (buffer only) 0 0.5 1 2 4 6
2.8 2.5 2.6 2.8 2.8 2.5 2.7
73 113 139 176 213 192 218
26.1 45.2 53.5 62.9 76.1 76.8 80.7
X X × X X × X
l0 s l0 s l0 s 105 10 s l0 s 10 S
208 90 [
80
70
? >
6O
~o 50
/
4O
30
0
015 1
1;
2.5
45
6J5
Total cyclophosphomide incubatron time (hours) Fig. 1. E f f e c t o f i n c u b a t i n g e y c l o p h o s p h a m i d e in b u f f e r at 3 0 ° C f o r increasing l e n g t h s of t i m e p r i o r to the a d d i t i o n of strain D3 cells for 0.5 h t o m o n i t o r i n d u c e d m i t o t i c r e c o m b i n a t i o n .
the forward mutation experiments using strain $288C, the petite (p-) m u t a n t isolate of strain D3 was more sensitive to the mutagenic activity of cyclophosphamide than was the parent (p*) strain. The red portion and the white portion of 53 sectored colonies induced by cyclophosphamide were isolated and tested for induced homozygosity of markers peripheral to the ade2 marker. In the homozygous red sectors 50 were also homozygous for histidine requirement, and in the white sectors the marker for cycloheximide sensitivity was homozygous in 51 of the isolates. Such results are expected if the red-sectored colonies are due to reciprocal mitotic crossing-over [30,53]. The few clones from red-sectored colonies which did not show the reciprocal homozygous phenotypes are most likely due to gene conversion or mutation at the ade2 locus [30,53]. The genetic activity of cyclophosphamide increased when the c o m p o u n d was incubated for increasing lengths of time in phosphate buffer without yeast cells (Table V). The increase in sector frequency can be more readily observed in Fig. 1. Sector frequency increased until approximately 2.5 h and then remained stable at that elevated level in the 4.5 and 6.5 h samples. Discussion
Cyclophosphamide induces genetic damage in several strains of S. cerevisiae without the requirement for mammalian metabolism to produce genetically active breakdown products. These findings are in contrast to most of the litera-
209 ture concerning the mutagenesis of this substance in a wide variety of test organisms, including bacteria [13,36,38] and yeast [8,14,15,41,42,43]. Tests for mutagenicity of cyclophosphamide using bacteria and yeast have been consistently negative unless mammalian metabolism was provided, either as a liver enzyme preparation [13] or in studies involving the intact animal [15,26,36, 40]. With one exception, a preliminary report from our laboratory [10], no strains of S. cerevisiae other than D4 have heretofore been thoroughly tested in vitro with cyclophosphamide. Our experience reported here confirms the fact that no genetic activity could be demonstrated for cyclophosphamide in strain D4 without some provision for mammalian metabolism [15,26,40,41--43]. The strain D4 used in the above investigations is respiratory-deficient (as is our own strain) and we considered the possibility that respiratory competence is necessary for in vitro cyclophosphamide activity. Accordingly we isolated petite clones from strains $288C and D3, anticipating that genetic tests in these isolates might be negative. Instead they showed a greater response than did the corresponding respiration-sufficient parent strains. It is worthy of note that plate tests without liver activation using yeast strain D4 (for gene conversion), strain $288C (for forward mutation) and strains $211 and S138 (both for reverse mutation) were all negative with cyclophosphamide [10]. Furthermore, plate tests using strain D4 with liver homogenate preparations were negative (D. Brusick, unpublished observations) in contrast to the numerous citations to positive results in comparable liquid suspension tests with strain D4. These results suggest that yeast plate tests, at least with some compounds, may be less sensitive to genetic damage than are comparable liquid suspension tests. Similar experience has been reported in bacterial plate tests with dimethyl- and diethylnitrosamine [-3]. On the other hand some compounds appear to be more sensitive in plate tests than in liquid suspension tests [3,32] {D. Brusick, unpublished observations). Strain $288C was used to measure forward mutation, while strains D3 and D5 measure with complete confidence only reciprocal mitotic crossing-over. Strain D4, in contrast, measures only gene conversion and from a superficial consideration of our data alone it would appear that without metabolic degradation cyclophosphamide can induce mutation and crossing-over but not gene conversion. On the other hand cyclophosphamide does indeed induce gene conversion in strain D4 when metabolic activation is included in the experimental method [15,26,40--43]. Furthermore, many of the sectored colonies observed in strains D3 and D5 that did not show the appropriate reciprocal products associated with mitotic crossingover were most probably due to gene conversion events. These studies suggest that cyclophosphamide does not preferentially induce one class of genetic event over another or that strain D4 is unique in some unknown respect. In our studies the respiration-deficient isolates of strains $288C and D3 were more sensitive to the genetic activity of cyclophosphamide than were the respiration-competent parent strains. Such sensitivity has been observed before [46, 47] and a possible reason for this sensitivity has been advanced [46]. This observation may deserve further attention for the development of strains with enhanced sensitivity to chemical mutagens. It should be noted, however, that the respiration-deficient phenotype may interfere with the expression of the
210
nuclear genetic effect under study. Such was the case in strain D3 in which it was necessary to use 8% instead of the usual 2% glucose [23] and to strictly follow the schedule for incubation of petri dishes and enumeration of sectored colonies. The red in the respiration-deficient isolate developed, then faded rapidly, but the color was quite stable in the respiration-competent strain D3. The canavanine-resistant phenotype was not affected by the respiration condition of the strain $288C cells. Experiments on the mutagenicity of cyclophosphamide were conducted at room temperature (23°C) and at 30°C. It was anticipated that elevating the treatment temperature would increase the mutagenicity either by accelerating the spontaneous degradation of cyclophosphamide or by enhancing the metabolism of the yeast cells to take up and distribute the c o m p o u n d more rapidly. Such increases were observed in experiments with strains $288C and D3; however, no differences were found in D4. A similar enhancement of genetic activity by elevation of the treatment temperature has been previously observed for chromosome aberrations in Vicia faba [31]. Consideration of the rather extensive literature describing the in vitro breakdc~,~n of cyclophosphamide [1,2,4,5,16,17,27,44] and consideration of our own mutagenicity data reported here leads us to believe that the compound is not active per se, but requires degradation, either spontaneously in aqueous solution or metabolically in the liver and perhaps other organs. An experiment was designed to observe more directly the possibility of spontaneous degradation of cyclophosphamide to form genetically active breakdown products. Aliquots of cyclophosphamide in buffer were incubated for increasing time intervals before strain D3 cells were added for an additional 0.5 h of incubation. If cyclophosphamide were active per se and not degraded to genetically active breakdown products, then the same frequency of sectored colonies would be expected in all treated samples because the yeast cells would all be exposed to the compound for the same length of time. If, however, the genetic activity were dependent on the accumulation over a period of time of active breakdown products, then later samples would show greater genetic activity than earlier samples. Our experiments demonstrated an increasing frequency of induced recombination from 0 to 2.5 h after which no further increase was apparent. Thus the in vitro genetic activity of cyclophosphamide for S. cerevisiae is dependent on spontaneously generated active breakdown products. It appears from early studies [4,5] and from more recent observations [16, 17] that the pathways of spontaneous degradation of cyclophosphamide differ from metabolic activation pathways and result in the formation of different end products in each case. The fact that some end products may be identical for both in vitro and in vivo processes is also noted [1]; however, it is possible that strain D4 is responsive only to end products formed by in vivo processes while the other yeast strains used can respond to end products formed in the spontaneous degradation in aqueous medium. These experiments raise questions about the role played by the spontaneously generated breakdown of cyclophosphamide in in vivo processes such as antineoplastic activity or genetic activity in mammals.
211
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