125
Mutation Research, 45 ( 1 9 7 7 ) 1 2 5 - - 1 3 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
MUTAGENIC AND EPIGENETIC INFLUENCE OF CAFFEINE ON THE FREQUENCIES OF UV-INDUCED OUABAIN-RESISTANT CHINESE HAMSTER CELLS *
C H I A - C H E N G C H A N G 1, C O N N I E PHILIPPS 1, J A M E S E. T R O S K O 1 and R O N W. H A R T 2
1 Department of Human Development, College of Human Medicine, Michigan State University, East Lansing, Mich. 48824 and 2Department of Radiology, Ohio State University, Columbus, Ohio (U.S.A.) (Received 12 April, 1977) ( A c c e p t e d 16 May, 1977)
Summary Caffeine, given as a post-treatment to UV-irradiated Chinese hamster cells in vitro, modified the frequency of induced mutations at the ouabain resistance locus. Mutation frequencies were increased when caffeine was added only for the DNA repair and mutation fixation period. When caffeine was added after the DNA repair and mutation fixation period, or immediately after DNA damage and for the entire repair and selection period, mutation frequencies were reduced. A hypothesis, given to explain both results, is that caffeine, by blocking a constitutive "error-free" postreplication repair process, allows an "errorprone" DNA repair process to produce many mutations. Moreover, caffeine, possibly by modifying C-AMP metabolism, causes a repression of induced mutations which, in effect, explains its anti-mutagenic and anti-carcinogenic properties.
Introduction Because of the potential role of somatic mutations in the carcinogenic [6,27, 50,52,55] and in the ageing [11,44,47,49] processes, it will be necessary to characterize the molecular basis for mutagenesis. The role of DNA repair mechanisms in mutagenesis has been firmly established in Escherichia coli [28]. The concepts of "error-free" and "error-prone" DNA repair have been created to differentiate those DNA repair processes which enzymatically render damaged DNA to its original condition from those which generate mutations [61]. * This work was supported by a National Cancer Institute R e s e a r c h Career D e v e l o p m e n t Award (CA 24085-05) to J.E.T. and a grant f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e (CA 13048-03).
126 Radman [36] had postulated an inducible postreplication repair enzyme ("SOS"-repair) to be responsible for mutagenesis in microbial systems. Subsequently, genetic [62] and biochemical [4~,43] evidence for such an inducible repair enzyme was reported. In mammalian cells, excision [37] and "postreplication", or "gap-filling" repair [10,19,29,30,31,51,54] mechanisms have been demonstrated. Maher and McCormick [32] have inferred that the excision repair mechanism in normal human cells is an "error-free" process, based on their report of higher induced mutation frequencies in fibroblasts of the excision repair deficient xeroderma pigmentosum (XP) syndrome than in normal fibroblasts. Higgins et al. [22] have postulated a model for the "post-replication" (replication) repair process, which, on the surface of things, would provide an error-free mechanism to bypass unexcised lesions in template DNA in mammalian cells. D'Ambrosio and Setlow [13] have reported observations which they interpret as evidence for an inducible repair system in Chinese hamster cells. Caffeine has been shown to inhibit a postreplication repair process in many mammalian [10,19,30,39,51,54] and some human cells [31], although it does not inhibit excision repair [37] or unscheduled DNA synthesis [9] in these cells. The literature is replete with a mass of seemingly contradictory observations on the biological consequences of caffeine posttreatment of physical and chemical carcinogen-treated mammalian cells. For example, Trosko and Chu [ 51] had shown that caffeine posttreatment of UV-irradiated Chinese hamster cells reduced the recovery of mutations, whereas others have shown that the frequency of mutations was increased over those not treated with caffeine [38]. Moreover, Donovan and DiPaolo [15] have shown an enhanced effect on the recovery of chemical cacinogen-induced transformed Syrian hamster cells, while Kakunaga [24] has shown an inhibitory effect of caffeine on 4-nitroquinoline-l-oxide induced transformed mouse cells. Also, caffeine has been reported to reduce the recovery of carcinogen-induced tumors in mouse skin [35,40,63]. In this report, we have designed experiments to help resolve the apparent conflicting results of carcinogen-exposed cells, posttreated with caffeine. Our results seem to indicate that (a) caffeine has multiple, but distinct and separable effects on UV-irradiated cells; (b) caffeine inhibits an error-free "postreplication" repair mechanism; (c) caffeine represses induced mutations after they were "fixed and expressed"; (d) there exists a causal relation between mutagenesis, in vitro transformation and in vivo tumorigenesis; and (e) there might exist a caffeine-insensitive, error-prone enzyme in Chinese hamster cells. Materials and methods
Cell culture An aneuploid cell line (V79), derived originally from the lung of a male Chinese hamster (Cricetulus griseus, 2n = 22), was used [17]. Cells were grown in modified Eagle's medium [16], supplemented with "non-essential" amino acids, 1 mM sodium pyruvate and 5% fetal calf serum. Cell survival To determine the survival fraction of cells treated with UV and caffeine, the
127 cells were plated in quadruplicate plates for a t t a c h m e n t for three and a half hours before medium change and UV irradiation. With the medium removed from the plates, the attached cells were exposed to ultraviolet light from a germicidal lamp (GE 15T8-15W) which was positioned to deliver a fluence of 10 ergs/mm2/sec (1 J/m2/sec). Various non-toxic concentrations of caffeine were presented in the medium at various times and in both survival and m u t a t i o n plates. The plating efficiencies were determined from the average percentage of survivors in the four plates. In vitro m u t a t i o n assay
The effects of caffeine on mutation were tested in an assay system involving the forward m u t a t i o n from ouabain sensitivity to resistance [48]. Cells were plated in 9 cm plates for attachment for 3.5 h before medium change and UVirradiation. Caffeine and the selective agent (ouabain, 1 mM; Sigma Chemical Company, St. Louis, Missouri) were added to the medium at various times and were present for different durations. Resistant colonies developed one week later. They were scored and isolated for further tests in ouabain (1 mM)-containing media. The survival plates were treated in the same manner except that no selective agent (ouabain) was added. The protocol for the ouabain resistance mutation assay was systematically characterized (paper in preparation). The optimum expression time (42--48 h after UV irradiation) and cell densities were used for the mutation experiments involving caffeine.
Results To characterize the effect of caffeine on our in vitro Chinese hamster mutation system, the experiments, summarized in Fig. 1 and Table 1, were performed. At all concentrations of caffeine used, there was no effect on survival, although at high concentrations of caffeine cell growth was slightly retarded and colony size was smaller. Caffeine did have a synergistic effect on survival of UV-irradiated cells as has been demonstrated by others [14,25,33,56]. Caffeine did n o t appear to affect the spontaneous frequency of ouabain resistant cells at 0.2 mM and 0.5 mM concentrations. The spontaneous mutation frequency seemed to be reduced at 0.9 mM concentration. The effect of the time at which caffeine was added to UV-irradiated cells on the recovery of UV-induced ouabain resistant cells is seen in Table 2. When caffeine was added to cells immediately after UV irradiation during the period for DNA repair and mutation expression, caffeine enhanced the frequency of UVinduced ouabain resistant colonies. When caffeine was present immediately after UV-irradiation for the entire postirradiation period, it appeared to reduce the frequency of UV-induced mutants. Caffeine significantly reduced the frequency of UV-induced mutants when it was added to cells after the completion of DNA repair and m u t a t i o n expression but for the remainder of the incubation period. It should also be noted that caffeine significantly affected cell survival only when it was present immediately after UV-irradiation (compare Expts 1 with 3 in Table 2).
128
100
\\
I0 %
, Co~ ne
Survivol
ffeine
30
50
75
I00
150
200
UV Fluence (ercJs/mm2)
Fig. 1. T h e e f f e c t of v a r i o u s c o n c e n t r a t i o n s of c a f f e i n e o n t h e ability of Chinese h a m s t e r cells t o f o r m colonies after UV-irradiation.
To refine further the differential effect of caffeine on UV-irradiated cells according to the time of posttreatment, the experiments in Table 3 and 4 were performed. Again, it seems that 0.5 mM caffeine, if present immediately after UV for the period of D N A repair and mutation expression period, will enhance UV-induced killing and mutation frequency. In Table 4, caffeine drastically reduced the recovery of ouabain-resistant mutants if UV-irradiated cells were
TABLE 1 S U R V I V A L A N D F R E Q U E N C Y OF S P O N T A N E O U S M U T A T I O N S FROM O U A B A I N S E N S I T I V I T Y TO RESISTANCE AFTER TREATMENT WITH CAFFEINE Caffeine a (mM)
% survival c
M u t a t i o n f r e q u e n c y per 106 s u r v i v o r s (No. of m u t a n t s )
0 b 0.2 b 0.5 b 0.9 b
100 111 108 120
4.8 4.8 4.0 1.2
a b c d
(10) (11) (9) ( 3) d
Cells w e r e p l a t e d in m e d i u m c o n t a i n i n g c a f f e i n e , and r e m a i n e d in it for e n t i r e i n c u b a t i o n p e r i o d . N o . of cells s e e d e d and plates (9 c m ) used: 30 X 105 ]30. Plating e f f i c i e n c y of t h e c o n t r o l , 70%. H i g h l y significant d e c r e a s e at P < 0.01 w h e n c o m p a r e d t o t h e c o n t r o l g r o u p o f n o n - c a f f e i n e t r e a t e d cells [45].
129 TABLE 2 SURVIVAL AND FREQUENCY OF MUTATIONS AFTER UV AND CAFFEINE TREATMENT Expt No.
UV fluence ( e r g s / m m 2)
FROM OUABAIN SENSITIVITY TO RESISTANCE
Caffeine (raM)
% survival d
Mutation frequency per 106 survivors
(No. of mutants) 1 a
0 70 70 70
f g h i
0 0 0.2 0.5
100 94 65 6.6
14 28 97 e 350 e
(46) (105) (296) (137)
2 b
0 70 70 70
f g h i
0 0 0.2 0.5
100 86 74 26
1.4 28 16 e 16
( 4) (82) (48) (22)
3 c
0 70 70 70
f g g g
0 0 0.2 0.5
100 82 94 90
5.0 (13) 18 (42) 6.0 e (16) 3 . 0 e ( 8)
a b c
C a f f e i n e w a s p r e s e n t d u r i n g t h e e x p r e s s i o n p e r i o d o n l y , i.e. f o r 4 4 h f o l l o w i n g U V i r r a d i a t i o n . C a f f e i n e w a s p r e s e n t f o r e n t i r e p o s t - i r r a d i a t l o n p e r i o d , i.e. f o r a p p r o x i m a t e l y 2 4 0 h. Caffeine was added 44 h after UV irradiation, and was present for the remainder of the incubation period. d P l a t i n g e f f i c i e n c y f o r c o n t r o l s : E x p t . 1 , 1 1 3 % ; E x p t . 2, 9 6 % ; E x p t . 3, 8 0 % . e H i g h l y s i g n i f i c a n t i n c r e a s e s o r d e c r e a s e s (P
Mutation Research, 45 ( 1 9 7 7 ) 1 2 5 - - 1 3 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
MUTAGENIC AND EPIGENETIC INFLUENCE OF CAFFEINE ON THE FREQUENCIES OF UV-INDUCED OUABAIN-RESISTANT CHINESE HAMSTER CELLS *
C H I A - C H E N G C H A N G 1, C O N N I E PHILIPPS 1, J A M E S E. T R O S K O 1 and R O N W. H A R T 2
1 Department of Human Development, College of Human Medicine, Michigan State University, East Lansing, Mich. 48824 and 2Department of Radiology, Ohio State University, Columbus, Ohio (U.S.A.) (Received 12 April, 1977) ( A c c e p t e d 16 May, 1977)
Summary Caffeine, given as a post-treatment to UV-irradiated Chinese hamster cells in vitro, modified the frequency of induced mutations at the ouabain resistance locus. Mutation frequencies were increased when caffeine was added only for the DNA repair and mutation fixation period. When caffeine was added after the DNA repair and mutation fixation period, or immediately after DNA damage and for the entire repair and selection period, mutation frequencies were reduced. A hypothesis, given to explain both results, is that caffeine, by blocking a constitutive "error-free" postreplication repair process, allows an "errorprone" DNA repair process to produce many mutations. Moreover, caffeine, possibly by modifying C-AMP metabolism, causes a repression of induced mutations which, in effect, explains its anti-mutagenic and anti-carcinogenic properties.
Introduction Because of the potential role of somatic mutations in the carcinogenic [6,27, 50,52,55] and in the ageing [11,44,47,49] processes, it will be necessary to characterize the molecular basis for mutagenesis. The role of DNA repair mechanisms in mutagenesis has been firmly established in Escherichia coli [28]. The concepts of "error-free" and "error-prone" DNA repair have been created to differentiate those DNA repair processes which enzymatically render damaged DNA to its original condition from those which generate mutations [61]. * This work was supported by a National Cancer Institute R e s e a r c h Career D e v e l o p m e n t Award (CA 24085-05) to J.E.T. and a grant f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e (CA 13048-03).
126 Radman [36] had postulated an inducible postreplication repair enzyme ("SOS"-repair) to be responsible for mutagenesis in microbial systems. Subsequently, genetic [62] and biochemical [4~,43] evidence for such an inducible repair enzyme was reported. In mammalian cells, excision [37] and "postreplication", or "gap-filling" repair [10,19,29,30,31,51,54] mechanisms have been demonstrated. Maher and McCormick [32] have inferred that the excision repair mechanism in normal human cells is an "error-free" process, based on their report of higher induced mutation frequencies in fibroblasts of the excision repair deficient xeroderma pigmentosum (XP) syndrome than in normal fibroblasts. Higgins et al. [22] have postulated a model for the "post-replication" (replication) repair process, which, on the surface of things, would provide an error-free mechanism to bypass unexcised lesions in template DNA in mammalian cells. D'Ambrosio and Setlow [13] have reported observations which they interpret as evidence for an inducible repair system in Chinese hamster cells. Caffeine has been shown to inhibit a postreplication repair process in many mammalian [10,19,30,39,51,54] and some human cells [31], although it does not inhibit excision repair [37] or unscheduled DNA synthesis [9] in these cells. The literature is replete with a mass of seemingly contradictory observations on the biological consequences of caffeine posttreatment of physical and chemical carcinogen-treated mammalian cells. For example, Trosko and Chu [ 51] had shown that caffeine posttreatment of UV-irradiated Chinese hamster cells reduced the recovery of mutations, whereas others have shown that the frequency of mutations was increased over those not treated with caffeine [38]. Moreover, Donovan and DiPaolo [15] have shown an enhanced effect on the recovery of chemical cacinogen-induced transformed Syrian hamster cells, while Kakunaga [24] has shown an inhibitory effect of caffeine on 4-nitroquinoline-l-oxide induced transformed mouse cells. Also, caffeine has been reported to reduce the recovery of carcinogen-induced tumors in mouse skin [35,40,63]. In this report, we have designed experiments to help resolve the apparent conflicting results of carcinogen-exposed cells, posttreated with caffeine. Our results seem to indicate that (a) caffeine has multiple, but distinct and separable effects on UV-irradiated cells; (b) caffeine inhibits an error-free "postreplication" repair mechanism; (c) caffeine represses induced mutations after they were "fixed and expressed"; (d) there exists a causal relation between mutagenesis, in vitro transformation and in vivo tumorigenesis; and (e) there might exist a caffeine-insensitive, error-prone enzyme in Chinese hamster cells. Materials and methods
Cell culture An aneuploid cell line (V79), derived originally from the lung of a male Chinese hamster (Cricetulus griseus, 2n = 22), was used [17]. Cells were grown in modified Eagle's medium [16], supplemented with "non-essential" amino acids, 1 mM sodium pyruvate and 5% fetal calf serum. Cell survival To determine the survival fraction of cells treated with UV and caffeine, the
127 cells were plated in quadruplicate plates for a t t a c h m e n t for three and a half hours before medium change and UV irradiation. With the medium removed from the plates, the attached cells were exposed to ultraviolet light from a germicidal lamp (GE 15T8-15W) which was positioned to deliver a fluence of 10 ergs/mm2/sec (1 J/m2/sec). Various non-toxic concentrations of caffeine were presented in the medium at various times and in both survival and m u t a t i o n plates. The plating efficiencies were determined from the average percentage of survivors in the four plates. In vitro m u t a t i o n assay
The effects of caffeine on mutation were tested in an assay system involving the forward m u t a t i o n from ouabain sensitivity to resistance [48]. Cells were plated in 9 cm plates for attachment for 3.5 h before medium change and UVirradiation. Caffeine and the selective agent (ouabain, 1 mM; Sigma Chemical Company, St. Louis, Missouri) were added to the medium at various times and were present for different durations. Resistant colonies developed one week later. They were scored and isolated for further tests in ouabain (1 mM)-containing media. The survival plates were treated in the same manner except that no selective agent (ouabain) was added. The protocol for the ouabain resistance mutation assay was systematically characterized (paper in preparation). The optimum expression time (42--48 h after UV irradiation) and cell densities were used for the mutation experiments involving caffeine.
Results To characterize the effect of caffeine on our in vitro Chinese hamster mutation system, the experiments, summarized in Fig. 1 and Table 1, were performed. At all concentrations of caffeine used, there was no effect on survival, although at high concentrations of caffeine cell growth was slightly retarded and colony size was smaller. Caffeine did have a synergistic effect on survival of UV-irradiated cells as has been demonstrated by others [14,25,33,56]. Caffeine did n o t appear to affect the spontaneous frequency of ouabain resistant cells at 0.2 mM and 0.5 mM concentrations. The spontaneous mutation frequency seemed to be reduced at 0.9 mM concentration. The effect of the time at which caffeine was added to UV-irradiated cells on the recovery of UV-induced ouabain resistant cells is seen in Table 2. When caffeine was added to cells immediately after UV irradiation during the period for DNA repair and mutation expression, caffeine enhanced the frequency of UVinduced ouabain resistant colonies. When caffeine was present immediately after UV-irradiation for the entire postirradiation period, it appeared to reduce the frequency of UV-induced mutants. Caffeine significantly reduced the frequency of UV-induced mutants when it was added to cells after the completion of DNA repair and m u t a t i o n expression but for the remainder of the incubation period. It should also be noted that caffeine significantly affected cell survival only when it was present immediately after UV-irradiation (compare Expts 1 with 3 in Table 2).
128
100
\\
I0 %
, Co~ ne
Survivol
ffeine
30
50
75
I00
150
200
UV Fluence (ercJs/mm2)
Fig. 1. T h e e f f e c t of v a r i o u s c o n c e n t r a t i o n s of c a f f e i n e o n t h e ability of Chinese h a m s t e r cells t o f o r m colonies after UV-irradiation.
To refine further the differential effect of caffeine on UV-irradiated cells according to the time of posttreatment, the experiments in Table 3 and 4 were performed. Again, it seems that 0.5 mM caffeine, if present immediately after UV for the period of D N A repair and mutation expression period, will enhance UV-induced killing and mutation frequency. In Table 4, caffeine drastically reduced the recovery of ouabain-resistant mutants if UV-irradiated cells were
TABLE 1 S U R V I V A L A N D F R E Q U E N C Y OF S P O N T A N E O U S M U T A T I O N S FROM O U A B A I N S E N S I T I V I T Y TO RESISTANCE AFTER TREATMENT WITH CAFFEINE Caffeine a (mM)
% survival c
M u t a t i o n f r e q u e n c y per 106 s u r v i v o r s (No. of m u t a n t s )
0 b 0.2 b 0.5 b 0.9 b
100 111 108 120
4.8 4.8 4.0 1.2
a b c d
(10) (11) (9) ( 3) d
Cells w e r e p l a t e d in m e d i u m c o n t a i n i n g c a f f e i n e , and r e m a i n e d in it for e n t i r e i n c u b a t i o n p e r i o d . N o . of cells s e e d e d and plates (9 c m ) used: 30 X 105 ]30. Plating e f f i c i e n c y of t h e c o n t r o l , 70%. H i g h l y significant d e c r e a s e at P < 0.01 w h e n c o m p a r e d t o t h e c o n t r o l g r o u p o f n o n - c a f f e i n e t r e a t e d cells [45].
129 TABLE 2 SURVIVAL AND FREQUENCY OF MUTATIONS AFTER UV AND CAFFEINE TREATMENT Expt No.
UV fluence ( e r g s / m m 2)
FROM OUABAIN SENSITIVITY TO RESISTANCE
Caffeine (raM)
% survival d
Mutation frequency per 106 survivors
(No. of mutants) 1 a
0 70 70 70
f g h i
0 0 0.2 0.5
100 94 65 6.6
14 28 97 e 350 e
(46) (105) (296) (137)
2 b
0 70 70 70
f g h i
0 0 0.2 0.5
100 86 74 26
1.4 28 16 e 16
( 4) (82) (48) (22)
3 c
0 70 70 70
f g g g
0 0 0.2 0.5
100 82 94 90
5.0 (13) 18 (42) 6.0 e (16) 3 . 0 e ( 8)
a b c
C a f f e i n e w a s p r e s e n t d u r i n g t h e e x p r e s s i o n p e r i o d o n l y , i.e. f o r 4 4 h f o l l o w i n g U V i r r a d i a t i o n . C a f f e i n e w a s p r e s e n t f o r e n t i r e p o s t - i r r a d i a t l o n p e r i o d , i.e. f o r a p p r o x i m a t e l y 2 4 0 h. Caffeine was added 44 h after UV irradiation, and was present for the remainder of the incubation period. d P l a t i n g e f f i c i e n c y f o r c o n t r o l s : E x p t . 1 , 1 1 3 % ; E x p t . 2, 9 6 % ; E x p t . 3, 8 0 % . e H i g h l y s i g n i f i c a n t i n c r e a s e s o r d e c r e a s e s (P
