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Well-marked strains of aspergillus for tests of environmental mutagens: Identification of induced mitotic recombination and mutation
147
Mutation Research, 38 (1976) 147--150 C) Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication GENETIC EFFECTS OF POTASSIUM DICHROMATE IN SCHIZOSA CCHA R OM YCES POMBE *
STEFANIA BONATTI, M. MEINI and A. ABBONDANDOLO Laboratorio di Mutagenesi e Differenziamento del C.N.R., Pisa (Italy) (Received May 21st, 1975) (Revision received August 19th, 1975) (Accepted August 29th, 1975)
As an air pollutant, chromium is normally present in the atmosphere of cities and non-urban areas, where its concentration has been measured [ 1 1 ] . During industrial processing of chromium and its compounds, chromate dust, particles of alloys or fumes may be dispersed in the air of industrial plants and may subsequently be inhaled by workers [9,10]. Investigations have been performed on the level of chromium in organic fluids of workers [4], as well as on the toxicity [ 9 ] , c y t o t o x i c i t y [6] and carcinogenicity [2] of chromium compounds. The relevant information a b o u t occurrence, use and biological activity in animals and man has been recently reviewed [3]. In view of the widely accepted correlation between carcinogenic and mutagenic activity of chemicals, it is of interest to ascertain whether chromium compounds are mutagenic. While this research was in progress, the mutagenicity of hexavalent chromium salts of Na, K and Ca was demonstrated in Escherichia coil [ 1 2 ] . Evidence is presented here that gene conversion is induced by K2Cr2 07 in the yeast Schizosaccharomyces pombe. Mutation induction in a forward mutation, 2 genes system [8] is also reported, although this data cannot be considered as definitive p r o o f of mutagenic activity. The wild-type, haploid strain of S. p o m b e 972, h- was used in forward mutation experiments. Mitotic gene conversion was studied on the heteroallelic, diploid strains ade 7, 50/150, ade 7, 50/151, ade 7, 50/152, ade 7, 50/2 75. Relative positions and map distances of the five involved alleles have been reported [7]. The effect of chromium was studied by treating, at room temperature, washed stationary phase cells with K2Cr207 freshly dissolved in sodium citrate/ citric acid 0.1 M buffer (pH 4.5) in which the c o m p o u n d is present as Cr2072ion. For mutation experiments, treated cells were washed and plated (200 viable cells per petri dish) on complete medium (YEL [8] ), where ade6 and ade7 mutants can be scored as purple colonies [8]. For gene conversion experi* Publication No.
1 2 9 f r o m the L a b o r a t o r i o di M u t a g e n e s i e D i f f e r e n z i a m e n t o del
C.N.R., Pisa, Italy.
148
30-
25-
i
:°I
/
%
,-_,
101
10 4
5 x 10 dt
K2Cr207
10 ~
(.uM)
F i g . 1. K 2 C r 2 O T - i n d u c e d g e n e c o n v e r s i o n i n f o u r h e t e r o a i l e l i c , d i p l o i d s t r a i n s o f S c h i z o s a c c h a r o m y c e s p o r n b e . S t r a i n ade7, 5 0 / 1 5 0 : y = 1 . 0 1 2 2 6 6 + 0 . 0 0 0 0 2 4 x ; F = 2 7 . 2 9 7 8 0 ; r = 0 . 9 3 3 9 0 . S t r a i n ade7, 5 0 / 151: y = 0 . 5 0 6 2 0 4 + 0 . 0 0 0 0 6 3 x ; F = 1 7 9 . 7 0 4 5 ; r = 0 . 9 4 8 6 0 . S t r a i n ade7, 5 0 / 1 5 2 : y = 2 . 1 2 1 0 9 7 + 0 . 0 0 0 0 8 5 x ; F = 2 7 7 . 6 7 8 8 ; r = 0 . 9 9 7 8 7 . S t r a i n ade7, 5 0 / 2 7 5 : y = 3 . 4 3 7 7 3 7 + 0 . 0 0 0 2 6 9 x ; F = 1 0 6 0 . 7 0 4 ; r = 0.99812.
For explanation
o f s y m b o l s a n d c a l c u l a t i o n s , see t e x t .
ments, cells were treated for 20 min with 102 to 105 pM potassium dichromate, and l 0 s to 2 X 106 cells per petri dish (depending on spontaneous frequency of conversion of each heteroallelic strain) were plated on minimal medium [8]. Diluted samples were plated on YEL for estimating cell viability. In a series of 10 experiments of forward mutation, 7 purple mutants were scored out of 480,054 colonies that survived after exposure of the haploid strain to 102 ~M K2Cr207 for 7 h. The average survival was 39.4%. No m u t a n t was found among 84,546 colonies grown from untreated cells. Spontaneous mutation frequency from white to purple in S. pombe had been previously estimated at 0.6 X 10 -7 on a cellular basis [8], and no purple colonies had been found in a sample of approx. 106 colonies [8]. The mutation frequency of the treated population (1.5 X 10 -s) is n o t significantly different [5] from that of the parallel control (<1.2 X 10-s). However, if the previous, larger population (approx. 106 cells) is taken as control, 7 mutants are sufficient to demonstrate mutation induction (P < 0.01) [5]. Although the last criterion is probably allowed in view of the remarkable stability of spontaneous mutation frequency in this genetic system, its questionableness is acknowledged.
149 Fig. 1 shows the kinetics of gene conversion induced by potassium dichromate on each of the four allelic combinations tested. Conversion frequencies were treated by linear regression analysis, according to the equation y -- a + b x , where y is the observed frequency of convertants and x the dose of potassium dichromate. The regression coefficient b is the slope of the linear relationship observed and represents the a m o u n t by which the induced frequency (y) increases for a unitary increase of the dose (x). The coefficient a represents the frequency of convertants at a dose x = 0. Three independent experiments were performed for each allelic combination, and the correlation coefficient r as well as the variance F was calculated. The correlation coefficient r is 1 when every increase of the dose (x) is associated with a proportional increase in the frequency of genetic effect (y) [1]. With all allelic combinations, a significant linear increase of gene conversion was observed. In summary, with the obvious limitations inherent with the use of sub-mammalian systems for the evaluation of genetic or carcinogenic risk for man, further studies on the potential hazards posed by chromium compounds seem desirable on the basis of the genetic activity demonstrated on yeast cells.
Acknowledgements Experiments were technically performed by G. Corti and A. Mazzaccaro. The help of S. Baroncelli in statistical analysis is acknowledged.
References 1 Crow, J.F. a n d M. Kimttra, A n i n t z o d u c t i o n t o p o p u l a t i o n genetics t h e o r y , H a r p e r a n d R o w , New York, 1970. 2 H u e p e r , W.C. a n d W.W. P a y n e , E x p e r i m e n t a l studies in m e t a l carcinogenesis, A r c h . Environ. Health, 5 ( 1 9 6 2 ) 445---462. 3 Iarc, M o n o g r a p h s o n t h e E v a l u a t i o n o f C a r c i n o g e n i c Risk o f Chemicals t o Man, 2, 1 0 0 - - 1 2 5 , Intern a t i o n a l A g e n c y f o r R e s e a r c h o n Cancer, L y o n , 1 9 7 3 . 4 I m b u s , H.R., J. C h o l a k , L.H. Miller a n d T. Sterling, B o r o n , c a d m i u m , chro*mium, a n d nickel in b l o o d a n d urine, A r c h . Environ. H e a l t h , 6 ( 1 9 6 3 ) 2 8 6 - - 2 9 5 . 5 K a s t e n b a u m , M.A. a n d K.O. B o w m a n , Tables f o r d e t e r m i n i n g t h e statistical significance of m u t a t i o n frequencies, M u t a t i o n Res., 9 ( 1 9 7 0 ) 5 2 7 - - 5 4 9 . 6 Levis, A.G., M. B u t t i g n o l a n d L. V e t t o r a t o . C h r o m i u m c y t o t o x i c effectS on m a m m a l i a n cells in vitro, A t t i Assoc. Genet. Its]., 2 0 ( 1 9 7 5 ) 9 - - 1 2 . 7 L o p r i e n o , N., R. Barale a n d S. Baroncelli, Genetic effects of caffeine, M u t a t i o n Res., 26 ( 1 9 7 4 ) 8 3 - 87. 8 L o p r i e n o , N., S. B o n a t t i , A. A b b o n d a n d o l o a n d R. Guglielminetti, The n a t u r e o f s p o n t a n e o u s m u t a t i o n d u r i n g vegetative g r o w t h in Schizosaccharomyces pombe, Molec, Gen. Genet., 1 0 4 ( 1 9 6 9 ) 4 0 - - 5 0 . 9 Pierce, J.O. a n d L.D. Scheel, T o x i c i t y o f alloys of c h r o m i u m , A r c h . Environ. Health, 1 0 ( 1 9 6 5 ) 8 7 0 - 876. 10 Samitz, M.H. a n d S. K a t z , P r o t e c t i o n a g a i n s t i n h a l a t i o n of c h r o m i c acid mist, Arch. E n v ~ o n . Health, 11 ( 1 9 6 5 ) 7 7 0 - - 7 7 2 . 11 S c h r o e d e r , H.A., A sensible l o o k a t air p o l l u t i o n b y metals, Arch. Environ. Health, 21 ( 1 9 7 0 ) 7 9 8 - 806. 12 Venitt, S. a n d L.S. Levy, M u t a g e n i c i t y o f c h r o m a t e s in b a c t e r i a a n d its relevance t o c h r o m a t e c a r c i n o genesis, N a t u r e , 2 6 0 ( 1 9 7 4 ) 493---495.
Mutation Research, 38 (1976) 147--150 C) Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication GENETIC EFFECTS OF POTASSIUM DICHROMATE IN SCHIZOSA CCHA R OM YCES POMBE *
STEFANIA BONATTI, M. MEINI and A. ABBONDANDOLO Laboratorio di Mutagenesi e Differenziamento del C.N.R., Pisa (Italy) (Received May 21st, 1975) (Revision received August 19th, 1975) (Accepted August 29th, 1975)
As an air pollutant, chromium is normally present in the atmosphere of cities and non-urban areas, where its concentration has been measured [ 1 1 ] . During industrial processing of chromium and its compounds, chromate dust, particles of alloys or fumes may be dispersed in the air of industrial plants and may subsequently be inhaled by workers [9,10]. Investigations have been performed on the level of chromium in organic fluids of workers [4], as well as on the toxicity [ 9 ] , c y t o t o x i c i t y [6] and carcinogenicity [2] of chromium compounds. The relevant information a b o u t occurrence, use and biological activity in animals and man has been recently reviewed [3]. In view of the widely accepted correlation between carcinogenic and mutagenic activity of chemicals, it is of interest to ascertain whether chromium compounds are mutagenic. While this research was in progress, the mutagenicity of hexavalent chromium salts of Na, K and Ca was demonstrated in Escherichia coil [ 1 2 ] . Evidence is presented here that gene conversion is induced by K2Cr2 07 in the yeast Schizosaccharomyces pombe. Mutation induction in a forward mutation, 2 genes system [8] is also reported, although this data cannot be considered as definitive p r o o f of mutagenic activity. The wild-type, haploid strain of S. p o m b e 972, h- was used in forward mutation experiments. Mitotic gene conversion was studied on the heteroallelic, diploid strains ade 7, 50/150, ade 7, 50/151, ade 7, 50/152, ade 7, 50/2 75. Relative positions and map distances of the five involved alleles have been reported [7]. The effect of chromium was studied by treating, at room temperature, washed stationary phase cells with K2Cr207 freshly dissolved in sodium citrate/ citric acid 0.1 M buffer (pH 4.5) in which the c o m p o u n d is present as Cr2072ion. For mutation experiments, treated cells were washed and plated (200 viable cells per petri dish) on complete medium (YEL [8] ), where ade6 and ade7 mutants can be scored as purple colonies [8]. For gene conversion experi* Publication No.
1 2 9 f r o m the L a b o r a t o r i o di M u t a g e n e s i e D i f f e r e n z i a m e n t o del
C.N.R., Pisa, Italy.
148
30-
25-
i
:°I
/
%
,-_,
101
10 4
5 x 10 dt
K2Cr207
10 ~
(.uM)
F i g . 1. K 2 C r 2 O T - i n d u c e d g e n e c o n v e r s i o n i n f o u r h e t e r o a i l e l i c , d i p l o i d s t r a i n s o f S c h i z o s a c c h a r o m y c e s p o r n b e . S t r a i n ade7, 5 0 / 1 5 0 : y = 1 . 0 1 2 2 6 6 + 0 . 0 0 0 0 2 4 x ; F = 2 7 . 2 9 7 8 0 ; r = 0 . 9 3 3 9 0 . S t r a i n ade7, 5 0 / 151: y = 0 . 5 0 6 2 0 4 + 0 . 0 0 0 0 6 3 x ; F = 1 7 9 . 7 0 4 5 ; r = 0 . 9 4 8 6 0 . S t r a i n ade7, 5 0 / 1 5 2 : y = 2 . 1 2 1 0 9 7 + 0 . 0 0 0 0 8 5 x ; F = 2 7 7 . 6 7 8 8 ; r = 0 . 9 9 7 8 7 . S t r a i n ade7, 5 0 / 2 7 5 : y = 3 . 4 3 7 7 3 7 + 0 . 0 0 0 2 6 9 x ; F = 1 0 6 0 . 7 0 4 ; r = 0.99812.
For explanation
o f s y m b o l s a n d c a l c u l a t i o n s , see t e x t .
ments, cells were treated for 20 min with 102 to 105 pM potassium dichromate, and l 0 s to 2 X 106 cells per petri dish (depending on spontaneous frequency of conversion of each heteroallelic strain) were plated on minimal medium [8]. Diluted samples were plated on YEL for estimating cell viability. In a series of 10 experiments of forward mutation, 7 purple mutants were scored out of 480,054 colonies that survived after exposure of the haploid strain to 102 ~M K2Cr207 for 7 h. The average survival was 39.4%. No m u t a n t was found among 84,546 colonies grown from untreated cells. Spontaneous mutation frequency from white to purple in S. pombe had been previously estimated at 0.6 X 10 -7 on a cellular basis [8], and no purple colonies had been found in a sample of approx. 106 colonies [8]. The mutation frequency of the treated population (1.5 X 10 -s) is n o t significantly different [5] from that of the parallel control (<1.2 X 10-s). However, if the previous, larger population (approx. 106 cells) is taken as control, 7 mutants are sufficient to demonstrate mutation induction (P < 0.01) [5]. Although the last criterion is probably allowed in view of the remarkable stability of spontaneous mutation frequency in this genetic system, its questionableness is acknowledged.
149 Fig. 1 shows the kinetics of gene conversion induced by potassium dichromate on each of the four allelic combinations tested. Conversion frequencies were treated by linear regression analysis, according to the equation y -- a + b x , where y is the observed frequency of convertants and x the dose of potassium dichromate. The regression coefficient b is the slope of the linear relationship observed and represents the a m o u n t by which the induced frequency (y) increases for a unitary increase of the dose (x). The coefficient a represents the frequency of convertants at a dose x = 0. Three independent experiments were performed for each allelic combination, and the correlation coefficient r as well as the variance F was calculated. The correlation coefficient r is 1 when every increase of the dose (x) is associated with a proportional increase in the frequency of genetic effect (y) [1]. With all allelic combinations, a significant linear increase of gene conversion was observed. In summary, with the obvious limitations inherent with the use of sub-mammalian systems for the evaluation of genetic or carcinogenic risk for man, further studies on the potential hazards posed by chromium compounds seem desirable on the basis of the genetic activity demonstrated on yeast cells.
Acknowledgements Experiments were technically performed by G. Corti and A. Mazzaccaro. The help of S. Baroncelli in statistical analysis is acknowledged.
References 1 Crow, J.F. a n d M. Kimttra, A n i n t z o d u c t i o n t o p o p u l a t i o n genetics t h e o r y , H a r p e r a n d R o w , New York, 1970. 2 H u e p e r , W.C. a n d W.W. P a y n e , E x p e r i m e n t a l studies in m e t a l carcinogenesis, A r c h . Environ. Health, 5 ( 1 9 6 2 ) 445---462. 3 Iarc, M o n o g r a p h s o n t h e E v a l u a t i o n o f C a r c i n o g e n i c Risk o f Chemicals t o Man, 2, 1 0 0 - - 1 2 5 , Intern a t i o n a l A g e n c y f o r R e s e a r c h o n Cancer, L y o n , 1 9 7 3 . 4 I m b u s , H.R., J. C h o l a k , L.H. Miller a n d T. Sterling, B o r o n , c a d m i u m , chro*mium, a n d nickel in b l o o d a n d urine, A r c h . Environ. H e a l t h , 6 ( 1 9 6 3 ) 2 8 6 - - 2 9 5 . 5 K a s t e n b a u m , M.A. a n d K.O. B o w m a n , Tables f o r d e t e r m i n i n g t h e statistical significance of m u t a t i o n frequencies, M u t a t i o n Res., 9 ( 1 9 7 0 ) 5 2 7 - - 5 4 9 . 6 Levis, A.G., M. B u t t i g n o l a n d L. V e t t o r a t o . C h r o m i u m c y t o t o x i c effectS on m a m m a l i a n cells in vitro, A t t i Assoc. Genet. Its]., 2 0 ( 1 9 7 5 ) 9 - - 1 2 . 7 L o p r i e n o , N., R. Barale a n d S. Baroncelli, Genetic effects of caffeine, M u t a t i o n Res., 26 ( 1 9 7 4 ) 8 3 - 87. 8 L o p r i e n o , N., S. B o n a t t i , A. A b b o n d a n d o l o a n d R. Guglielminetti, The n a t u r e o f s p o n t a n e o u s m u t a t i o n d u r i n g vegetative g r o w t h in Schizosaccharomyces pombe, Molec, Gen. Genet., 1 0 4 ( 1 9 6 9 ) 4 0 - - 5 0 . 9 Pierce, J.O. a n d L.D. Scheel, T o x i c i t y o f alloys of c h r o m i u m , A r c h . Environ. Health, 1 0 ( 1 9 6 5 ) 8 7 0 - 876. 10 Samitz, M.H. a n d S. K a t z , P r o t e c t i o n a g a i n s t i n h a l a t i o n of c h r o m i c acid mist, Arch. E n v ~ o n . Health, 11 ( 1 9 6 5 ) 7 7 0 - - 7 7 2 . 11 S c h r o e d e r , H.A., A sensible l o o k a t air p o l l u t i o n b y metals, Arch. Environ. Health, 21 ( 1 9 7 0 ) 7 9 8 - 806. 12 Venitt, S. a n d L.S. Levy, M u t a g e n i c i t y o f c h r o m a t e s in b a c t e r i a a n d its relevance t o c h r o m a t e c a r c i n o genesis, N a t u r e , 2 6 0 ( 1 9 7 4 ) 493---495.