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Mutation Research, 54 (1978) 193--196 © Elsevier/North-Holland Biomedical Press

COMPARISON OF TOXICITY AND MUTAGENICITY OF BUTYL METHANESULFONATE AMONG HUMAN LYMPHOBLAST LINES

SAUL A. SLAPIKOFF 1, BARBARA M. ANDON : and WILLIAM G. THILLY 2

1 Department of Biology, Tufts University, Medford, Mass. 02155, and 2 Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.) (Received 5 January 1978) (Revision received 24 April 1978) (Accepted 3 May 1978)

Summary The toxic and mutagenic effects of butyl methanesulfonate (BMS) were compared among four diploid human lymphoblast lines, MIT-2, WI-L2, MGL8B-2 and GM 130. The toxic and mutagenic effects of 24-h exposure to BMS were similar for the MIT-2, WI-L2 and MGL8B-2 lines, while the GM 130 line was strikingly resistant to the toxic and mutagenic effects of BMS.

The purine analogues, 8-azaguanine, 8-azahypoxanthine and 6-thioguanine (6TG) are toxic to most mammalian cells. Exceptions are rare variants that either lack hypoxanthine--guanine phosphoribosyl transferase (HGPRT) activity [1] or lack the ability to transport these purine analogues [3]. Thus, these analogues can be used as selective agents in gene-locus assays that test for mutation induced by physical or chemical agents [1]. Using 6TG as a selective agent in combination with a soft-agar overlay technique that permits the cloning of non-attachment dependent cells [6], this laboratory has developed a quantitative assay for estimating the amount of gene-locus mutation induced by physical and chemical agents in diploid human lymphoblasts [2,4,5,8]. Since all of our previous studies have utilized a single human lymphoblast line (designated MIT-2) originally derived from a female mononucleosis patient, we could not assess the extent to which our results were typical of the response

Abbreviations: BMS, butyl methanesulfonate~ HGPRT, hypoxanthine--guanine phosphoribosyl

transferase; MNNG, methylnitronitrosoguanidine: MNU, methylnitrosourea~ 6TG, 6-thioguanine.

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to mutagens of lymphoblasts derived from an "average" person. We thus have undertaken to compare the results obtained using MIT-2 cells as the target cells for mutagens with the results obtained using established lines of diploid lymphoblasts obtained from other individuals. Our criterion for selecting a particular lymphoblast line has been that it must have at least a 5% plating efficiency using the soft-agar overlay technique that our assay requires. Briefly, unstirred suspension cultures, 50--400 ml at 4 X l 0 s cells/ml in RPMI 1640 supplemented with 14% fetal bovine serum, were treated with various concentrations of mutagen. After 24 h of treatment, cells were resuspended in fresh medium without mutagen. Thereafter the cultures were counted daily (Coulter Electronics, Inc., Hialeah, Florida) and diluted to 3.5--4 X l 0 s cells/ml daily. Cell survival was determined immediately after removal of the test compound by a soft-agar overlay technique utilizing human fibroblasts as a feeder layer. After sufficient time ( > 1 2 days) for expression of the mutant p h e n o t y p e (HGPRT-) [4], the mutant fraction was determined by soft-agar plating of the cultures in the presence and the absence of 6GT (10 ~g/ml) using as a feeder layer 6TG-resistant human fibroblasts derived from a Lesch--Nyhan patient. That this time was indeed sufficient for pkenotypic expression was demonstrated by the constancy of the observed mutant fraction in three separate platings on days 13, 16, 17 and 19 after treatment. In addition to the MIT-2 lymphoblast line, which is a clonal derivative of the PGLC-33 line isolated from a female mononucleosis patient by Dr. Phillip R. Glade, University of Miami, we have used in this study: GM 130, a line established by Dr. A.D. Bloom, College of Physicians and Surgeons of Columbia University, from lymphocytes of a normal male and purchased from the Institute for Medical Research, Camden, New Jersey; MGL8B-2, a gift from the Genetics Unit, Massachusetts General Hospital, is a clonal derivative (containing a trisomy 15 which arose spontaneously during culture) of line MGL8 which was isolated from a male donor; and WI-L2, a line originally established at the Wistar Institute, Philadelphia, from lymphocytes of a male donor with hereditary spherocytosis, was a gift of Dr. A.D. Bloom. The lines grow as spinner cultures in continuous exponential phase in RPMI 1640 supplemented with 14% fetal bovine serum with the following approximate doubling times: MIT-2, 20 h; GM 130, 30 h; MGL8B-2, 20 h; and WI-L2, 13 h. The plating efficiencies of MIT-2 and WI-L2 are 25--50% and GM 130 and MGL8B-2 lines 5--10% over a confluent fibroblast feeder layer. We chose to use BMS in these experiments since previous studies in this laboratory comparing the toxicity and mutagenicity of methyl methanesulfonate, ethyl methanesulfonate, propyl methanesulfonate, and butyl methanesulfonate on MIT-2 indicated that BMS was more mutagenic at a lower toxicity than the other alkyl methanesulfonates tested. BMS has also been reported to have bactericidal and mutagenic action toward E. coli [9]. The toxic and mutagenic effects of a 24-h exposure to BMS on cultures of the four human lymphoblast lines are shown in Fig. 1. Most striking is the resistance of GM 130 lymphoblasts to the toxic and mutagenic effects of BMS. At 3 mM BMS, the surviving fraction was 0.014 for MGL8B-2 and 0.00014 for MIT-2 and WI-L2. Gm 130 lymphoblasts also demonstrated resistance to the mutagenic effects of BMS at this concentration. There is some variation in the

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Fig. I. T o x i c i t y and m u t a g e n i e i t y of BMS to h u m a n lYn',phoblast lines. R e l a t i v e survival a f t e r 24-h t r e a t m e n t (open symbols) as determined by soft-agar plating. The m u t a n t fraction (closed symbols) was d e t e r m i n e d by octuplieate ( M I T - 2 a n d W I - L 2 ) o r d o d e c u p l i c a t e (GM 1 3 0 a n d M G L S B - 2 ) p l a t i n g s o n d a y s 13, 16, 17 a n d 19 after treatment allowing p h e n o t y p i c e x p r e s s i o n o f the H G P R T - state and is obtained b y dividing the plating efficiency in the presence of 6 T G b y the plating efficiency in the absence of 6TG. The lines drawn through the m u t a n t fraction s y m b o l s are least s q u a r e linear regressions of the data Points. The d a t a are e x p e r s s e d as m e a n ± s.e.m. Absence of e r r o r s bars indicate that ± s.e.m, for that data point are within the s y m b o l size. o, M I T - 2 ; o, M G L 8 B - 2 ; v GM 1 3 0 ; ~, W I - L 2 .

surviving fractions following BMS treatment of the other three lines; the mutant fractions induced in MIT-2 and WI-L2 by 24-h exposure to BMS are similar, while the mutant fraction induced in MGL8B-2 is somewhat lower. Line GM 130 is obviously an interesting exception. It is not only resistant to the toxic and mutagenic effects of BMS at the concentrations tested; it is also highly resistant to toxic and mutagenic effects of methylnitrosourea {MNU) and methylnitronitrosoguanidine (MNNG) at concentrations that are highly toxic and mutagenic to MIT-2 [4,8] (GM 130 data not shown). Further, since lines GM 130 and MIT-2 were treated at the same time with the same stock solution o f BMS, the resistance of line GM 130 to the toxic and mutagenic effects of BMS is not due to possible mishandling of the treatment solution.

196 It is possible that the failure to observed the induction of HGPRT- mutants in line GM 130 by BMS is due to a failure to provide sufficient time for expression of the m u t a n t phenotype. In previous studies with MIT-2 [4] we have found that as m a n y as 14 generations post-treatment are required for the stable expression of the m u t a n t phenotype. Days 13, 16, 17 and 19 after treatment, which were the days on which platings for measurement of m u t a n t fractions were made, correspond to 10, 13, 14 and 15 generations respectively for line GM 130. The presence of an 8-fold higher level of HGPRT in GM 130 or a greater stability of HGPRT to proteolysis in GM 130 could conceivably have caused us to fail to observe mutation induced in GM 130. We have, however, observed the presence of MIT-2 cells heritably resistant to toxic effects of these alkylating agents [4,8]. The basis for the resistance of GM 130 and the resistant subpopulations of MIT-2 to the toxic and possibly to the mutagenic effects of these alkylating agents remains to be explained. We would like to conclude that WI-L2, MIT-2 and MGL8B-2 represent a kind of average behavior of diploid human lymphoblast lines treated with BMS. However, since the sample of human diploid lymphoblasts tested is small, we have some reservations in drawing this conclusion. We consider this work to be another step in the direction of testing the possible importance of interspecies, intertissue, and interpersonal variation in sensitivity to chemical mutations. Such data are, of course, necessary to the process of devising accurate means to differentiate between chemicals which are potentially hazardous or non-hazardous to humans. We wish to thank Henry Hoppe IV, John DeLuca, and Bruce Penman for their helpful discussions. References 1 C h u , E . H . Y . , a n d H . V . Mailing, M a m m a l i a n cell g e n e t i c s , II. C h e m i c a l i n d u c t i o n o f specific l o c u s m u t a t i o n s in C h i n e s e h a m s t e r cells in v i t r o , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 6 1 ( 1 9 6 8 ) 1 3 0 6 - - 1 3 1 2 . 2 D e L u c a , J . G . , D . A . K a d e n , J . J . K r o l e w s k i , T . R . S k o p e k a n d W.G. T h i l l y , C o m p a r a t i v e m u t a g e n i c i t y o f I C R - 1 9 1 t o S. t y p h i m u r i u m a n d d i p l o i d h u m a n l y m p h o b l a s t s , M u t a t i o n Res., 4 6 ( 1 9 7 7 ) 1 1 - - 1 8 . 3 GiUin, F . D . , D . J . R o u f a , A . L . B e a u d e t a n d C.T. C a s k e y , 8 - A z a g u a n i n e r e s i s t a n c e in m a m m a l i a n cells. I. H y p o x a n t h i n e - - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e , G e n e t i c s , 72 ( 1 9 7 2 ) 2 3 9 - - 2 5 2 . 4 P e n m a n , B.W., a n d W.G. T h i l l y , C o n c e n t r a t i o n - d e p e n d e n t m u t a t i o n of d i p l o i d h u m a n l y m p h o b l a s t s b y m e t h y l n i t r o n i t r o s o g u a n i d i n e : t h e i m p o r t a n c e o f p h e n o t y p i c lag, S o r e . Cell G e n e t . , 2 ( 1 9 7 6 ) 3 2 5 - - 3 3 0 . 5 P e n m a n , B.W., M.V. W o n g a n d W.G. T h i l l y , M u t a g e n i c i t y o f 5 - h a l o d e o x y u r i d i n e s t o d i p l o i d h u m a n l y m p h o b l a s t s , Life Sci., 19 ( 1 9 7 6 ) 5 6 3 - - 5 6 8 . 6 S a t o , K., R.S. Slesinski a n d J.W. L i t t l e f i e l d , C h e m i c a l m u t a g e n e s i s a t t h e p h o s p h o r i b o s y l t r a n s f e r a s c l o c u s in c u l t u r e d h u m a n l y m p h o b l a s t s , P r o c . Na~l. A c a d . Sci. ( U . S . A . ) , 6 9 ( 1 9 7 2 ) 1 2 4 4 - - 1 2 4 8 . 7 S z y b a l s k i , W., a n d E . H . S z y b a l s k a , D r u g s e n s i t i v i t y as a g e n e t i c m a r k e r f o r h u m a n cell lines, U n i v . M i c h . M e d . Bull., 2 8 ( 1 9 6 2 ) 2 7 7 - - 2 9 3 . 8 T h i l l y , W . G . , J . G . D e L u c a , H . H o p p e IV a n d B.W. P e n m a n , M u t a t i o n o f h u m a n l y m p h o h l a s t s b y m e t h y l nitrosourea, Chem.-Biol. Interact., 15 (1976) 33--50. 9 T u r t o c z k y , I., a n d L. E h r e n b e r g , R e a c t i o n r a t e s a n d b i o l o g i c a l a c t i o n o f a l k y l a t i n g a g e n t s , p r e l i m i n a r y r e p o r t o n b a c t e r i c i d a l a n d m u t a g e n i c a c t i o n in E. coli, M u t a t i o n R e s . , 8 ( 1 9 6 9 ) 2 2 9 - - 2 3 8 .

Comparison of toxicity and mutagenicity of butyl methanesulfonate among human lymphoblast lines.

193 Mutation Research, 54 (1978) 193--196 © Elsevier/North-Holland Biomedical Press COMPARISON OF TOXICITY AND MUTAGENICITY OF BUTYL METHANESULFONAT...
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