Recombinogenicity and mutagenicity of saccharin in Saccharomyces cerevisiae

215

Mutation Research, 67 (1979) 215-219

© Elsevier/North-Holland Biomedical Press

RECOMBINOGENICITY AND MUTAGENICITY OF SACCHARIN IN S A C C H A R OM Y C E S C E R E V I S I A E

CAROLW MOORE and ANNSCHMICK Departments of Biology, and Radiation Biology and Biophysics, The Universzty of Rochester, Rochester, N Y (U S.A )

(Received 23 August 1978) (Revision received 1 February 1979) (Accepted 27 February 1979)

Summary Diploid yeast grown in the presence of a commercial lot of saccharin exhibited reproducible, dose-dependent increases in intergenic and intragenic recombination, and mutation. Cells grew to nearly the same titer in media w i t h o u t saccharin and containing 2 or 20 mg saccharin/ml, although cell viability was somewhat reduced in saccharin-containing media. At the high test dose of 100 mg/ml, titers and cell viability were more markedly lowered. Differences between this study and previous (negative) tests o f saccharin in yeast are described.

Many chemicals which are carcinogenic and mutagenic are also recombinogenic. Mutational and recombinational events can be measured very accurately in S a c c h a r o m y c e s cerevisiae, unlike systems in procaryotes, humans, other higher or most eucaryotic organisms. To determine if yeast grown in the presence of saccharin exhibit increases in mitotic intergenic and intragenic recombination, and mutation, the sweetener was tested in numerous experiments in this laboratory in a sensitive genetic system designed to detect several types of genetic events. A comprehensive study of saccharin effects of these types has n o t been reported previously. Materials and methods Following our decision to study yeast grown in liquid media containing saccharin, it seemed important in our experimental designs that cultures witho u t saccharin and with the lowest saccharin doses tested should grow at equal rates and undergo equal numbers of cell divisions, to avoid bias in favor of recombinant or revertant cells. After several modifications of routine types of

216 growth media for yeast were tried, a nonsynthetic complete m e d m m lacking dextrose was adopted. For the experiments described m this commumcatlon, equal aliquots of a fresh, stationary phase, YPAD-grown culture of diploid strain CM-1293 were inoculated into 1% yeast extract, 2% peptone, 0.16 mg/ml ademne sulfate (YPA). Saccharin (Lot No. 65C-0129, Sigma, sodmm salt) had been added t() YPA at the test concentrations. Following incubation with aeration at 30 ~ for 60--65 h, cells were washed, counted in a hemacytometer, and several different dilutions of cells plated on synthetic complete and selective media [7,18]. Colonies were counted and recombinant and revertant classes scored following incubation of plates at 30 ° for periods of tm~e appropmate for each type of media (2--6 days). Results and Discussion YPA w i t h o u t saccharin and containing up to 20 mg saccharin per ml regularly supported growth of diploid yeast to nearly the same titer for a particular strain. Titers and cell viability following growth in 100 mg/ml and 250 mg/ml were lowered. Often morphologies of cells growing in these high doses were abnormal, especially in 250 mg/ml. At this highest dose, cultures were also clustered and partly sporulated, a fact whmh could account for high frequencies of recombinant and revertant cells in such cultures; this led us to eliminate presentation of those genetic data here. Spore formation was never indicated in diploid cultures m 2, 20 and 100 mg/ml saccharin. Enhanced mutation frequencies in planned expemments employing haploid cells grown in the presence of saccharin would corroborate our genetic data from diploids. Table 1 summarizes typical dose-dependent frequencies of both classes of mitotic recombination studied in one stram. Intergenic exchanges between homologous chromosomes in diploid organisms entail breakage and degradation of DNA strands and formation of hybrid DNA, result m homozygosity of markers located on the same chromosomal arm distal to the exchange point, are primarily reciprocal, and are signalled unequivocally in a diploid bearing the two heteroalleles ade2-119 and ade2-40 by the formation of twin-sectored, pink-red colonies [17,18]. Mitotic crossing-over in such a strain grown in the presence of the very low dose of 2 mg/ml was always higher than in the same strain grown w i t h o u t saccharin; at higher doses of 20 and 100 mg/ml, 16- and 22-fold increases were typically observed. The production of other classes of aberrant colonies signalling a variety of other genetic events such as deletions, aneuploidy, point mutations and mitotic gene conversion [17,19] were also dose-dependent. Following growth in 2, 20 and 100 mg/ml of the same saccharin, strain D81 [20,7], which monitors crossing-over on a different chromosome than CM-1293, also yielded reproducible, dose-dependent increases in frequencies of mitotic intergenic recombination. Induction of mitotic intragenic recombination, caused by many mutagens and occuring primarily nonreciprocally by a process called gene conversion, was monitored using two pairs of heteroalleles. Reproducible, dose-dependent factors of increase in tryptophan-nonrequiring colomes from the heteroallelic trp5-12/trp5-27 [6], tryptophan-requlring diploid [16,19], CM-1293, ranged

217 TABLE

1

FREQUENCIES OF GENETIC EVENTS G R O W T H IN S A C C H A R I N - C O N T A I N I N G Events monitored

I N S a c c h a r o m y c e s celevtstae S T R A I N LIQUID MEDIA

CM-1293

FOLLOWING

C o n c e n t r a t i o n (rag s a c c h a r m / m l Y P A )

Cell tlter (cells/ml YPA) Sur~xvlng fraction (vlabflxty)

0

2

20

100

2.1 v 1 0 7 [ 1 0 0 _+ 9 % ]

1 7 v 107 74 ~ 8%

1.6 Y 1 0 7 61 ~ 8%

3 0 ~ 10 ° 24 ± 10%

Mitotic lntergenlc reconlblllatlon (crossovers), other aberrant colomes on s u r v i v i n g cells Sectored, red-pink 2,6 + 0 2 3 3 0 . 0 +- 6 Red 0 9 ± 0.43 14.5 ÷ 3.2 Pink 0.9 ~ 0,39 2.0 e 0.2

nonselective medium, per 103 4 2 . 1 ~_ 4 . 4 37 7 ÷ 6.2 10 9 ± 3.0

53 ± 10 31 ± 5 1 9.8 + 2 8

Sectored, other thanred-pmk Total aberrant

1 5 ± 1,0 5 9 +_ 2 , 1 3

2 1 +_ 0 . 3 5 48 6 ± 7.9

1.8 t 92 5 +

0.4 9

59 ± 2.4 99.7 t 12

CAN1

2 1 _~ 0 4

4 . 0 _+ 0 . 9

11.0 +

1 8

820

Intragenlc recombination (mitotic gene convertants) T R P 5 p e r 105 s u r w v m g c e l l s 6 7 -+ 0.9 CYC1 p e r 1 0 4 s u r v i v i n g c e l l s 4 . 9 _+ 1 . 5 Reverse mutation

(nlltotle gene revertants) 1 2 ÷ 0.5

IlV+ p e r 1 0 7 s u r v i v i n g c e l l s

15.2 t 2.5 8 . 8 _+ 3 . 0

85+_10

166 ~ lS 3 0 . 1 +~ 6

9.2±

3

155

+- 1 0

88

± 21 ± 10

37

+

78

S e v e r a l h a p l o x d s t r a t u s b e a r i n g ade2-40, ade2-119, trp5-12, t r p 5 - 2 7 a n d H v l - 9 2 w e r e k i n d l y d o n a t e d b y Dr F K Z l m m e r m a n n T h e s e m a r k e r s are t h e s a m e as t h o s e i n c o r p o r a t e d m t h e w i d e l y - u s e d d x p l o l d D 7 [ 1 9 ] . B y c o n v e n t i o n a l g e n e t m t e e h m q u e s o f c r o s s i n g , s p o r u l a t l o n and d i s s e c t i o n , m e l o t m s e g r e g a n t s w e r e obtained whmh contained the desired genettc and fermentation markers, and other characteristics desired for q u a n t i t a t i v e s t u d m s m v o l ~ l n g s e v e r a l t y p e s o f g r o w t h m e d i a . T w o o f t h e s e , C M 1 0 6 9 - 4 0 a n d C M 1 2 3 2 - 1 0 5 , w e r e u s e d t o c o n s t r u c t d o p l o t d s t r a t a C M - 1 2 9 3 b e a r i n g h e t e r o a l l e l e s a d c 2 - 4 0 / a d e 2 119. trp 5 - 1 2 / t r p b - 2 7 , a n d c y e l - 4 5 ~ e y e 1 - 1 3 1 , a n d h o m o a l l e l e s z l v l . 9 2 / z l v l - 9 2 , t h e s t r a t a w a s also m a d e h e t e r o z y g o u s at t h e c a n l ( e a n a ~ a m n e ) l o c u s R e p r o d u c i b l e , d o s e - d e p e n d e n t i n c r e a s e s w e r e o b t a i n e d m 1 0 experiments with CM-1293 and a slmdar diploid strata, CM-1194 [7]

from 2 to 25. Prototrophic colonies growing on selective medium containing lactate [10] momtored CYC1 intragenic recombinants arising from heteroallelic cyc1-45/cyc1-131 diploid cells. Defects in the structural gene encoding iso-l-cytochrome c in these mutant strains [10,11] render them unable to utilize lactate as the sole carbon source and form large colonies on lactate-containing synthetic medium. Growth of strain CM-1293 in the presence of 2, 20 and 100 mg/ml saccharin yielded 1.8 to 18 times more CYC1 convertants than growth without saccharin in the medium. Frequencies of both TRP5 and CYC1 prototrophic gene convertants summarized in Table 1 include both gene convertants and revertants, but the latter occur at frequencies several orders of magnitude lower than convertants, and thus are negligible. Saccharin increased about 30-fold the incidence of gene reversion (Table 1), the third type of genetic event examined. Cell populations exposed to the lowest and highest test doses were 4-fold different. Isoleucine-nonrequiring colonies in a strain homozygous ilv1-92/dv1-92 results from true reverse mutation and allele nonspecific suppressor mutations [3,14,19]. This study to determine if saccharin toxicologically affects growing yeast cells involved different test conditions than the two previous (negative) tests of saccharin in yeast, using yeast strain D3. We grew cells in media containing

218 saccharin to stationary phase, while D3 tests exposed nongrowing cells to saccharin in buffer for up to a few hours [8,9]. Inability to detect toxicity or mitotic crossing-over following exposure of nongrowing D3 cells to up to 50 mg/ml saccharin [9], a dose higher than doses in which the growing cells we tested exhibited mitotic crossing-over and some cytotoxicity, could reflect lack of or low penetrability of c o m p o u n d through the think wall characteristic of yeast cells. D3 and CM-1293 are both diploid and should respond similarly. D3 monitors intergenic recombinatmn and contains a different pair of ade genotypic markers for its detection than CM-1293; the latter contains markers to monitor additional classes of genetm events as well, but such markers do not affect how the strata responds to saccharin or other potentially carcmogemc compounds. In addition to the very different saccharin-exposure conditions, our experiments differ from D3 tests in the media employed to visualize sectors resulting from mitotic crossing-over, and in the saccharin preparations utilized. Since mutagenic activities or other toxic indicators of different saccharin lots vary [ 2,4,5,12], impuritms in the saccharin lot used in CM-1293 experiments could differ somewhat from those in the D3 test preparation. Most likely, however, our design exposing growing cells to saccharin in media in contrast to brief exposures of nongrowing cells in buffer, is the most important difference between our experimental designs and previous negative saccharin tests using yeast [8,9]. The reproducible dose effects observed in these and other (unpublished) experiments in this laboratory clearly establish that saccharin preparations can be recombinogemc and mutagenic. The lot of saccharin (No. 65C-0129, Sigma) used m our experiments with growing yeast is not mutagenlc in the Salmonella/ Ames assay [13]. The organic solvent soluble impurities (10--15 ppm) recently extracted from No. 65C-0129 are mutagenic for Salmonella typhimurium TA1538, but only when they are very highly concentrated [13]. Thus, it is not surprising that the reagent or commercial (impure) saccharin containing only small amounts of only 10--15 ppm of these organic solvent soluble impuritms was n o t mutagenic for Salmonella. It is perhaps surprising that the same lot is n o t only mutagenic, but also recombinogenic, in yeast (this communication; unpublished results), and suggests greater sensitivity of the yeast test conditions described in this communication. The system should be rather sensitive for the detection of weak mutagens. Lot No. 65C-0129 (Sigma) is similar to $1022 (Sherwm-Wllhams) which induced bladder cancer in rats m two generations [ 1]. Both commercial saccharin ($1022) and saccharin purified from the same lot did not cause mutagenlc or other genetic alterations in several short-term tests [9], including the mitotic recombination test in yeast D3 and the widely-used Salmonella/Ames test; the same impure and partmlly-purified saccharm, on the other hand, induced equivalent frequencms of sister-chromatld exchanges m human and Chinese hamster cells [15], suggesting the partially-purified saccharin, which still contains water-soluble impurities, causes weak effects in vitro. Tests of saccharin purified from lot No. 65C-0129 on yeast CM-1293 would determine if recombinogenic and mutagenic properties are attributable to impurities or to saccharin per se, but the u n f o r t u n a t e impossibility of extracting all water-soluble

219

impunties from this lot -- similar to other Maumee-produced saccharins, including S1022 [5,12,13] -- prevent such tests. Experiments designed to quantitatively test commercml and purifmd saccharin produced by a different process are in progress. Acknowledgements This study was supported in part by ACS grant VC-190, EHSC grant ES 01247, NSF grant BMS75-13172 and NIH grant GM12702, and has been designated UR (DOE) Report No. 3490-1563. References 1 A r n o l d D L., S.M C h a r b o n n e a u , C.A M o o d i e a n d I.C. M u n r o , L o n g - t e r m t o x i c i t y s t u d y w i t h ot o l u e n e - s u l f o n a m ~ d e a n d s a c c h a r i n , S o c i e t y of T o x m o l o g y , 1 6 t h A n n u a l Meeting, T o r o n t o , C a n a d a ( 1 9 7 7 ) A b s t r a c t No 78. 2 B a t z m g e r , R . P , S L. Ou a n d E B u c d l n g . S a c c h a r i n and o t h e r s w e e t e n e r s M u t a g e m c p r o p e r t i e s , Science, 1 9 8 ( 1 9 7 7 ) 9 4 4 - - 9 4 6 . 3 G u n d e l a c h , E , S u p p r e s s o r s t u d i e s on d v l m u t a n t s o f S a c c h a r o m y c e s cerevzsme, M u t a t i o n Res., 20 (1973) 25--33. 4 K r a m e r s , P . G . N . , T h e m u t a g e n t c l t y o f s a c c h a n n , M u t a t i o n Res., 32 ( 1 9 7 5 ) 8 1 - - 9 2 . 5 K r a m e r s , P G.N , M u t a g e m c x t y o f s a c c h a n n in D r o s o p h i l a . T h e possible role o f c o n t a m i n a n t s , M u t a t tlon Res , 56 ( 1 9 7 7 ) 1 6 3 - - 1 6 7 . 6 M a n n e y , T . R , A c t i o n of a s u p e r - s u p p r e s s o r in y e a s t in r e l a t i o n to allehc m a p p i n g a n d c o m p l e m e n t a t i o n , G e n e t i c s , 50 ( 1 9 7 4 ) 1 0 9 - - 1 2 1 . 7 M o o r e , C.W , B l e o m y c I n - m d u c e d m u t a t i o n and r e c o m b i n a t i o n in S a c c h a r o m y c e s cerevlsiae, M u t a t i o n Res , 58 ( 1 9 7 8 ) 4 1 - - 4 9 . 8 Newell, G W , a n d W.A. Maxwell, S t u d y of M u t a g e n i c E f f e c t s o f S a c c h a r i n ( i n s o l u b l e ) , Nataonal T e c h m c a l I n f o r m a t i o n Service, S p n n g h e l d , Va., ( 1 9 7 2 ) . 9 Office of T e c h n o l o g y A s s e s s m e n t , Congress o f the U n i t e d States, A p p e n d i x II, S h o r t - t e r m Tests, in: Cancer Testing Technology and Sacchann, October 1977 10 S h e r m a n , F,, J W. S t e w a r t , M J a c k s o n , R.A. G i l m o r e a n d J.H. P a r k e r , M u t a n t s of y e a s t d e f e c t i v e in l s o - l - c y t o c h r o m e c, G e n e t i c s , 77 ( 1 9 7 4 ) 2 5 5 - - 2 8 4 . 11 S t e w a r t , J.W., F. S h e r m a n , N . A . S h i p m a n a n d M. J a c k s o n , I d e n t i f i c a t i o n a n d m u t a t i o n a l r e l o c a t i o n o f t h e A U G c o d o n i n i t i a t i n g t r a n s l o c a t I o n of t i s o - l - c y t o c h r o m e c in yeast, J Biol C h e m . , 246 ( 1 9 7 1 ) 7429--7445. 12 S t o l t z , D R,, B Stavric, R Klassen, R.D. Bendall a n d J Craig, T h e m u t a g e m c i t y of s a c c h a r i n i m p u r i ties, I. D e t e c t i o n of m u t a g e n i c a c t i v i t y , J. E n v i r o n . P a t h o l . T o x l c o l . , 1 ( 1 9 7 7 ) 1 3 9 - - 1 4 6 13 S t o l t z , D.R , B. S t a v n c , R. Klassen a n d R D. Bendall, p e r s o n a l c o m m u n i c a t i o n . 14 T h u r l a u x , P., M. Miner, A . M . A . t e n Berge a n d F,K. Z l m m e r m a n n , G e n e t i c fine s t r U c t u r e a n d f u n c t i o n m u t a n t s a t t h e ~lvl gene l o c u s o f S a c c h a r o m y c e s cereviszae, Mol G e n G e n e t . , 1 1 2 ( 1 9 7 1 ) 6 0 - - 7 2 . 15 Wolff, S., a n d B. R o d i n , S a c c h a n n - i n d u c e d sister c h r o m a t l d e x c h a n g e s in Chinese h a m s t e r a n d h u m a n cells, S c i e n c e , 2 0 0 ( 1 9 7 8 ) 5 4 3 - - 5 4 5 16 Z l m m e r m a n n , F . K . , I n d u c t i o n o f m i t o t i c gene c o n v e r s m n b y m u t a g e n s , M u t a t i o n Res., 11 ( 1 9 7 1 ) 327--337. 17 Z u n m e r m a n n , F . K . , A y e a s t strain for w s u a l s c r e e n i n g for t h e t w o r e c i p r o c a l p r o d u c t s o f m i t o t i c crossing o v e r , M u t a t i o n Res., 21 ( 1 9 7 3 ) 2 6 3 - - 2 6 9 . 18 Z l m m e r m a n n , F . K . , P r o c e d u r e s used m t h e i n d u c t i o n of m i t o t i c r e c o m b i n a t i o n a n d m u t a t i o n in t h e y e a s t S a c c h a r o m y e e s cerev~s~ae, M u t a t i o n Res., 31 ( 1 9 7 5 ) 7 1 - - 8 6 . 19 Z l m m e r m a n n , F . K . , R. K e r n a n d H. R a s e n b e r g e r , A y e a s t strain for s i m u l t a n e o u s d e t e c t i o n of i n d u c e d mltotac crossing over, m i t o t i c gene c o n v e r s i o n a n d r e v e r s e m u t a t a o n , M u t a t i o n R e s , 28 ( 1 9 7 5 ) 3 8 1 - 388. 20 S l m m e r m a n n , F . K . a n d B K. Vlg, M u t a g e n s p e c i f i c i t y in t h e r e d u c t i o n of m i t o t m c r o s s i n g - o v e r m S a c c h a r o m y c e s cerevlszae, Mol. G e n . G e n e t . , 139 ( 1 9 7 5 ) 2 5 5 - - 2 6 8 .