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Induction of sister-chromatid exchanges in a rat-liver cell line with chemical carcinogens
Mutation Research, 91 (1981) 47-50 (cO Elsevier/North-Holland Biomedical Press
47
INDUCTION OF SISTER-CHROMATID EXCHANGES IN A RAT-LIVER CELL LINE WITH CHEMICAL CARCINOGENS
ANTHONY L. MEYER and BRIAN J. DEAN
Shell Research Limited, Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent ME9 8A G (Great Britain) (Accepted 1 September 1980)
In the search for new techniques to detect potential genotoxic substances we have reported the initiation of an epithelial-like rat-liver cell line (RL1) and its subsequent use in a short-term in vitro chromosome assay [1]. Dose-related increases in chromosome damage were observed after treatment with several promutagens/procarcinogens, indicating that RLI cells had retained significant metabolising enzyme activity, even after many passages in in vitro culture. The use of this cell line in mutagen testing thus precludes the addition of microsome preparations or metabolising feeder layers which are pre-requisites in many short-term assay systems. These observations have prompted the present study into the induction of sisterchromatid exchanges (SCEs) in RLI cells by the direct-acting alkylating agent, methyl nitronitrosoguanidine (Koch-Light Laboratories Ltd.) and the indirectacting polycyclic hydrocarbon, 7,12-dimethylbenz[a]anthracene (Sigma Chemicals). We present the preliminary findings in this report. Materials and methods Slide cultures were prepared by seeding 7.5 x 104 RLI cells in 0.5 ml of Eagles Minimal Essential Medium (MEM) supplemented with 10°70 foetal calf serum (Flow) onto glass microscope slides in 90-mm culture dishes. The dishes were incubated at 37°C in a 5°7o CO2 atmosphere for 2 h to allow cell attachment and then 15 ml of culture medium was added to each dish. After 24 h incubation, the medium was removed and replaced with fresh medium containing both 15 ~M bromodeoxyuridine (BUdR) (Sigma Chemicals) and the appropriate concentration of test compound dissolved in dimethylsulphoxide (DMSO). After a further 22 h (equivalent to 2 rounds of cell division), Colcemid (Flow) was added to each culture at a final concentration of 0.3 #g/ml. 2 h later the slides were washed in Hanks' balanced salt solution (Wellcome), subjected to hypotonic treatment (0.56°7o potassium chloride) for 7 min and then fixed in methanol/acetic acid (3:1) for 30 min. Air-dried preparations were processed by the fluorochrome plus Giemsa (FPG) technique of Perry and Wolff [4] to visualise the unifilar and bifilar BUdR substitution in the sister chromatids. All cultures were maintained in
48 38 36 34 32 30 E
==
28 26 24 22 20 18 16 14 12 r
I
I
I
I
I
.01
.05
.1
.2
.3
.4
Concentration of MNNG (//g/ml)
Fig. 1. Linear dose-related increase in SCE induction in RLI cells after treatment with increasing concentrations of M N N G . 38 36 34 32 3o
E
28
w
26
~
24
E
22
~
20 18 16 14 12
0.0001
I 0.0005
i 0.001
i
i
i
i
i
0.005
0.01
0.05
0.1
0.5
1.0
Concentration DMBA (/~g/ml)
Fig. 2. L o g - l i n e a r d o s e - r e l a t e d increase in S C E i n d u c t i o n in RL] cells a f t e r t r e a t m e n t with increasing concentrations of DMBA.
49
the dark to minimise photolysis o f DNA incorporated BUdR and thus background SCEs [2]. Finally the slides were coded and scored by 2 independent observers. Results and discussion Table 1 lists the mean number of SCEs counted in 20 RL1 metaphases per dose following 24-h exposures to increasing concentrations of MNNG and DMBA ranging from 0.01 to 0.4 #g/ml and 0.0001 to 1.0 #g/ml resp. Above these concentrations cell replication was inhibited to a point where insufficient second replication-cycle cells could be found for SCE determination. We have shown previously [1] that a concentration o f 0.25 #g/ml of both MNNG and DMBA when applied to RL1 cells for 24 h is the lowest dose capable o f inducing a significant increase in chromosome aberrations. However, SCE induction in RL~ cells treated under identical conditions occurs at lower concentrations of both compounds. This is seen most markedly with cells treated with DMBA, where a concentration as low as 1 nanogram per ml increases SCE frequency above control values (Table 1). These results confirm other observations that SCEs are a far more sensitive indicator of chemically induced DNA damage than are chromosomal aberrations both in vitro [5] and in vivo [3]. Our results show a dose-related increase in SCE induction which is a linear function of M N N G concentration (Fig. 1). However, it is significant that SCE induction is a log-linear function o f DMBA concentration (Fig. 2), since we have observed similar effects in RLl cells when scoring for chromosome damage after treatment with increasing concentrations of TABLE 1 I N D U C T I O N OF SISTER-CHROMATID EXCHANGES 1N RAT-LIVER (RLj) CELLS WITH C H E M I C A L CARCINOGENS Compound
Concentration (ug/ml)
Mean number of SCEs per metaphase _+ S.E.
Mean number of SCEs per chromosome
MNNG
0 0.01 0.05 0.1 0.2 0.4
15.10 16.45 18.53 20.22 24.40 33.20
_+ 0.87 +_ 0.75 +_ 0.67 _+_ 0.99 + 1.22 _+ 1.36
0.34 0.37 0.42 0.46 0.55 0.75
DMBA
0 0.0001 0.0005 0.001 0.005 0.01 0.05 0.1 0.5 1.0
13.56 14.00 13.06 21.40 23.08 25.90 29.70 31.00 35.60 34.40
+ 0.44 +__ 0.62 +_ 0.77 + 1.12 + 1.32 + 1.09 _+ 1.27 _+ 1.26 _+ 1.96 + 1.67
0.31 0.32 0.30 0.49 0.52 0.59 0.68 0.70 0.81 0.78
50 D M B A [1]. These findings w o u l d suggest that at critical c o n c e n t r a t i o n s o f D M B A one or m o r e o f the residual e n z y m e systems b e c o m e saturated, thus affecting the g e n e r a t i o n o f genotoxic intermediates. A l t h o u g h a t h o r o u g h study o f SCE i n d u c t i o n with a p p r o p r i a t e groups of genotoxic a n d c o n t r o l chemicals is clearly needed, the data reported here indicate that the rat-liver cell line ( R L 9 m a y prove a quick, inexpensive a n d sensitive a d d i t i o n to existing s h o r t - t e r m screening procedures. REFERENCES 1 Dean, B.J., and G. Hodson-Walker, An in vitro chromosome assay using cultured rat-liver cells, Mutation Res., 64 (1979) 329-337. 2 lkushima, T., and S. Wolff, Sister chromatid exchanges induced by light flashes to 5bromodeoxyuridine and 5-iododeoxyuridine substituted Chinese hamster chromosomes, Exp. Cell Res., 87 (1974) 15-19. 3 Nakanishi, Y., and E.L. Schneider, In vivo sister-chromatid exchange: A sensitive measure of DNA damage, Mutation Res., 60 (1979) 329-337. 4 Perry, P'., and S. Wolff, New Giemsa method for the differential staining of sister chromatids, Nature (London), 251 (1974) 156-158. 5 Solomon, E., and M. Bobrow, Sister chromatid exchanges - A sensitive assay of agents damaging human chromosomes, Mutation Res., 30 (1975) 273-278.
47
INDUCTION OF SISTER-CHROMATID EXCHANGES IN A RAT-LIVER CELL LINE WITH CHEMICAL CARCINOGENS
ANTHONY L. MEYER and BRIAN J. DEAN
Shell Research Limited, Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent ME9 8A G (Great Britain) (Accepted 1 September 1980)
In the search for new techniques to detect potential genotoxic substances we have reported the initiation of an epithelial-like rat-liver cell line (RL1) and its subsequent use in a short-term in vitro chromosome assay [1]. Dose-related increases in chromosome damage were observed after treatment with several promutagens/procarcinogens, indicating that RLI cells had retained significant metabolising enzyme activity, even after many passages in in vitro culture. The use of this cell line in mutagen testing thus precludes the addition of microsome preparations or metabolising feeder layers which are pre-requisites in many short-term assay systems. These observations have prompted the present study into the induction of sisterchromatid exchanges (SCEs) in RLI cells by the direct-acting alkylating agent, methyl nitronitrosoguanidine (Koch-Light Laboratories Ltd.) and the indirectacting polycyclic hydrocarbon, 7,12-dimethylbenz[a]anthracene (Sigma Chemicals). We present the preliminary findings in this report. Materials and methods Slide cultures were prepared by seeding 7.5 x 104 RLI cells in 0.5 ml of Eagles Minimal Essential Medium (MEM) supplemented with 10°70 foetal calf serum (Flow) onto glass microscope slides in 90-mm culture dishes. The dishes were incubated at 37°C in a 5°7o CO2 atmosphere for 2 h to allow cell attachment and then 15 ml of culture medium was added to each dish. After 24 h incubation, the medium was removed and replaced with fresh medium containing both 15 ~M bromodeoxyuridine (BUdR) (Sigma Chemicals) and the appropriate concentration of test compound dissolved in dimethylsulphoxide (DMSO). After a further 22 h (equivalent to 2 rounds of cell division), Colcemid (Flow) was added to each culture at a final concentration of 0.3 #g/ml. 2 h later the slides were washed in Hanks' balanced salt solution (Wellcome), subjected to hypotonic treatment (0.56°7o potassium chloride) for 7 min and then fixed in methanol/acetic acid (3:1) for 30 min. Air-dried preparations were processed by the fluorochrome plus Giemsa (FPG) technique of Perry and Wolff [4] to visualise the unifilar and bifilar BUdR substitution in the sister chromatids. All cultures were maintained in
48 38 36 34 32 30 E
==
28 26 24 22 20 18 16 14 12 r
I
I
I
I
I
.01
.05
.1
.2
.3
.4
Concentration of MNNG (//g/ml)
Fig. 1. Linear dose-related increase in SCE induction in RLI cells after treatment with increasing concentrations of M N N G . 38 36 34 32 3o
E
28
w
26
~
24
E
22
~
20 18 16 14 12
0.0001
I 0.0005
i 0.001
i
i
i
i
i
0.005
0.01
0.05
0.1
0.5
1.0
Concentration DMBA (/~g/ml)
Fig. 2. L o g - l i n e a r d o s e - r e l a t e d increase in S C E i n d u c t i o n in RL] cells a f t e r t r e a t m e n t with increasing concentrations of DMBA.
49
the dark to minimise photolysis o f DNA incorporated BUdR and thus background SCEs [2]. Finally the slides were coded and scored by 2 independent observers. Results and discussion Table 1 lists the mean number of SCEs counted in 20 RL1 metaphases per dose following 24-h exposures to increasing concentrations of MNNG and DMBA ranging from 0.01 to 0.4 #g/ml and 0.0001 to 1.0 #g/ml resp. Above these concentrations cell replication was inhibited to a point where insufficient second replication-cycle cells could be found for SCE determination. We have shown previously [1] that a concentration o f 0.25 #g/ml of both MNNG and DMBA when applied to RL1 cells for 24 h is the lowest dose capable o f inducing a significant increase in chromosome aberrations. However, SCE induction in RL~ cells treated under identical conditions occurs at lower concentrations of both compounds. This is seen most markedly with cells treated with DMBA, where a concentration as low as 1 nanogram per ml increases SCE frequency above control values (Table 1). These results confirm other observations that SCEs are a far more sensitive indicator of chemically induced DNA damage than are chromosomal aberrations both in vitro [5] and in vivo [3]. Our results show a dose-related increase in SCE induction which is a linear function of M N N G concentration (Fig. 1). However, it is significant that SCE induction is a log-linear function o f DMBA concentration (Fig. 2), since we have observed similar effects in RLl cells when scoring for chromosome damage after treatment with increasing concentrations of TABLE 1 I N D U C T I O N OF SISTER-CHROMATID EXCHANGES 1N RAT-LIVER (RLj) CELLS WITH C H E M I C A L CARCINOGENS Compound
Concentration (ug/ml)
Mean number of SCEs per metaphase _+ S.E.
Mean number of SCEs per chromosome
MNNG
0 0.01 0.05 0.1 0.2 0.4
15.10 16.45 18.53 20.22 24.40 33.20
_+ 0.87 +_ 0.75 +_ 0.67 _+_ 0.99 + 1.22 _+ 1.36
0.34 0.37 0.42 0.46 0.55 0.75
DMBA
0 0.0001 0.0005 0.001 0.005 0.01 0.05 0.1 0.5 1.0
13.56 14.00 13.06 21.40 23.08 25.90 29.70 31.00 35.60 34.40
+ 0.44 +__ 0.62 +_ 0.77 + 1.12 + 1.32 + 1.09 _+ 1.27 _+ 1.26 _+ 1.96 + 1.67
0.31 0.32 0.30 0.49 0.52 0.59 0.68 0.70 0.81 0.78
50 D M B A [1]. These findings w o u l d suggest that at critical c o n c e n t r a t i o n s o f D M B A one or m o r e o f the residual e n z y m e systems b e c o m e saturated, thus affecting the g e n e r a t i o n o f genotoxic intermediates. A l t h o u g h a t h o r o u g h study o f SCE i n d u c t i o n with a p p r o p r i a t e groups of genotoxic a n d c o n t r o l chemicals is clearly needed, the data reported here indicate that the rat-liver cell line ( R L 9 m a y prove a quick, inexpensive a n d sensitive a d d i t i o n to existing s h o r t - t e r m screening procedures. REFERENCES 1 Dean, B.J., and G. Hodson-Walker, An in vitro chromosome assay using cultured rat-liver cells, Mutation Res., 64 (1979) 329-337. 2 lkushima, T., and S. Wolff, Sister chromatid exchanges induced by light flashes to 5bromodeoxyuridine and 5-iododeoxyuridine substituted Chinese hamster chromosomes, Exp. Cell Res., 87 (1974) 15-19. 3 Nakanishi, Y., and E.L. Schneider, In vivo sister-chromatid exchange: A sensitive measure of DNA damage, Mutation Res., 60 (1979) 329-337. 4 Perry, P'., and S. Wolff, New Giemsa method for the differential staining of sister chromatids, Nature (London), 251 (1974) 156-158. 5 Solomon, E., and M. Bobrow, Sister chromatid exchanges - A sensitive assay of agents damaging human chromosomes, Mutation Res., 30 (1975) 273-278.