Mutation Research, 283 (1992) 75-81 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
75
MUTLET 0704
Cytogenetic and molecular characterization of the mutagenicity of chlorambucil in V79 cells Giinter Speit, Walter Menz, Christiane R6scheisen and Beate K6berle Abteilung Klinische Genetik, Universitiit Ulm, D-7900 Ulm, Germany (Received 30 March 1992) (Revision received 15 May 1992) (Accepted 20 May 1992)
Keywords: V79 cells; Chlorambucil; Repair inhibitors; Chromosome damage; Gene mutation; Southern blot; Polymerase chain reaction
Summary Chlorambucil (CBC) is used as a chemotherapeutic agent and immunosuppressant. Recently, it could be shown that CBC is considerably more effective than radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes. We therefore reinvestigated the in vitro genotoxic effects of CBC in V79 cells and characterized induced sister-chromatid exchanges (SCEs), chromosome aberrations and gene mutations by means of cytogenetic and molecular methods. CBC effectively induced chromosome aberrations and SCEs in a dose-dependent manner. The chromosome aberrations found after a 14-h treatment were mainly chromatid-type aberrations. 3-Aminobenzamide (3AB) did not influence the incidence of CBC-induced SCEs and chromosome aberrations. Combined treatment with CBC and caffeine (CAF) strongly increased the frequency of aberrations, but had no effect on the yield of SCEs. CAF at lower concentrations enhanced the production of chromatid breaks and exchange figures while higher concentrations (10 -3 M) caused multiple breaks and pulverised mitoses. Mutations at the hprt locus were induced in a narrow range of CBC concentrations (10 5 M - 2 × 10 -5 M) and the mutagenic effect was accompanied by strong cytotoxicity. The CBC-induced gene mutation frequency was not increased after CAF treatment. The molecular analysis of CBC-induced mutations by Southern hybridization and PCR demonstrated that CBC predominantly produced small alterations but not deletions or gross structural alterations in the hprt gene of V79 cells. For the first time, these results reveal striking differences in the mutagenic action of an alkylating agent in cultivated cells compared to germ-line cells at the molecular level.
Chlorambucil (CBC) is used as a chemotherapeutic agent and immunosuppressant. This substance, which is a nitrogen mustard derivative
Correspondence: Dr. Giinter Speit, Universit~it Ulm, Abteilung Klinische Genetik, Postfach 4066, D-7900 Ulm, Germany.
and a bifunctional alkylating agent, has been reported to induce gene mutations, chromosome a b e r r a t i o n s and sister-chromatid exchanges (SCEs) in various genotoxicity short-term test systems (Stevenson and Patel, 1973; Littlefield et al., 1979; Singh and Gupta, 1983; Athanasiou and Arzimanoglou, 1986). Recently, it could be shown that CBC is considerably more effective than
76
radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes (Russell et al., 1989; Rinchik et al., 1990). Only few chemicals which give a positive response in the specific locus test (SLT) have been analysed at the molecular level. As CBC produced an unusual stage-response pattern and an unexpectedly high yield of deletions in the SLT, we investigated whether CBC shows an unusual mutagenic response in vitro as well. Previous studies have shown that a broad spectrum of mutations ranging from point mutations through large deletions can be detected with the H P R T test in V79 ceils (Thacker and Ganesh, 1989; Thacker et al., 1990; K6berle and Speit, 1991). By means of cytogenetic and molecular methods we now characterised CBC-induced gene mutations in the in vitro H P R T test in relation to other genetic endpoints. As the induction of chromosome aberrations and lethality by alkylating agents and nitrogen mustard could be enhanced by DNA repair inhibitors like 3-aminobenzamide and caffeine (Lau and Pardee, 1982; Das et al., 1984; Jan et al., 1986), we furthermore studied the effect of inhibited repair on CBC-induced mutations. Materials and methods
The experiments were performed with a clonal derivative of the V79 Chinese hamster cell line.
Ceils were cultivated in minimal essential medium (MEM) with Earle's salts supplemented with 10% foetal calf serum and antibiotics in a humidified incubator at 37°C with 5% CO 2 at a pH of 7.2. Mutagen treatment was performed about 24 h after the start of the experimental subcultures. The test substances used were chlorambucil (CBC), 3-aminobenzamide (3AB) and caffeine (CAF). They were all purchased from Sigma (Munich, Germany). For the H P R T gene mutation test, about 5 × 106 cells were treated in each experiment for 2 h with CBC alone or for 14 h with CBC in the absence or presence of caffeine. The treated cultures were transferred as needed during the expression period (7 days). 6-Thioguanine-resistant colonies were analysed in 5 replicate petri dishes with 2 × 105 cells each. Colonies from independent cultures were isolated for the molecular analysis. Survival (relative plating efficiency) was determined by plating 200 cells into 4 replicate petri dishes at the end of mutagen treatment (PE 1) and at the end of the expression period (PE 2). The methods for D N A isolation, Southern analysis and multiplex polymerase chain reaction (PCR) analysis, were exactly the same as described in a recent paper (K6berle et al., 1991). For Southern analysis, high-molecular-weight DNA was digested with either EcoRI or BgllI and hybridised with a [32p]dCTP nick-translated hprt cDNA probe ( p H P T 12). The same DNA samples were used for PCR reactions. Exons 2-9
TABLE 1 SCE F R E Q U E N C I E S I N D U C E D BY CBC AND T H E I N F L U E N C E OF 3AB AND CAF Treatment
SCEs/cell + SEM
(CBC)
Protocol 1 a
Control 10 -7 M 2 x 10 -7 5)
75
MUTLET 0704
Cytogenetic and molecular characterization of the mutagenicity of chlorambucil in V79 cells Giinter Speit, Walter Menz, Christiane R6scheisen and Beate K6berle Abteilung Klinische Genetik, Universitiit Ulm, D-7900 Ulm, Germany (Received 30 March 1992) (Revision received 15 May 1992) (Accepted 20 May 1992)
Keywords: V79 cells; Chlorambucil; Repair inhibitors; Chromosome damage; Gene mutation; Southern blot; Polymerase chain reaction
Summary Chlorambucil (CBC) is used as a chemotherapeutic agent and immunosuppressant. Recently, it could be shown that CBC is considerably more effective than radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes. We therefore reinvestigated the in vitro genotoxic effects of CBC in V79 cells and characterized induced sister-chromatid exchanges (SCEs), chromosome aberrations and gene mutations by means of cytogenetic and molecular methods. CBC effectively induced chromosome aberrations and SCEs in a dose-dependent manner. The chromosome aberrations found after a 14-h treatment were mainly chromatid-type aberrations. 3-Aminobenzamide (3AB) did not influence the incidence of CBC-induced SCEs and chromosome aberrations. Combined treatment with CBC and caffeine (CAF) strongly increased the frequency of aberrations, but had no effect on the yield of SCEs. CAF at lower concentrations enhanced the production of chromatid breaks and exchange figures while higher concentrations (10 -3 M) caused multiple breaks and pulverised mitoses. Mutations at the hprt locus were induced in a narrow range of CBC concentrations (10 5 M - 2 × 10 -5 M) and the mutagenic effect was accompanied by strong cytotoxicity. The CBC-induced gene mutation frequency was not increased after CAF treatment. The molecular analysis of CBC-induced mutations by Southern hybridization and PCR demonstrated that CBC predominantly produced small alterations but not deletions or gross structural alterations in the hprt gene of V79 cells. For the first time, these results reveal striking differences in the mutagenic action of an alkylating agent in cultivated cells compared to germ-line cells at the molecular level.
Chlorambucil (CBC) is used as a chemotherapeutic agent and immunosuppressant. This substance, which is a nitrogen mustard derivative
Correspondence: Dr. Giinter Speit, Universit~it Ulm, Abteilung Klinische Genetik, Postfach 4066, D-7900 Ulm, Germany.
and a bifunctional alkylating agent, has been reported to induce gene mutations, chromosome a b e r r a t i o n s and sister-chromatid exchanges (SCEs) in various genotoxicity short-term test systems (Stevenson and Patel, 1973; Littlefield et al., 1979; Singh and Gupta, 1983; Athanasiou and Arzimanoglou, 1986). Recently, it could be shown that CBC is considerably more effective than
76
radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes (Russell et al., 1989; Rinchik et al., 1990). Only few chemicals which give a positive response in the specific locus test (SLT) have been analysed at the molecular level. As CBC produced an unusual stage-response pattern and an unexpectedly high yield of deletions in the SLT, we investigated whether CBC shows an unusual mutagenic response in vitro as well. Previous studies have shown that a broad spectrum of mutations ranging from point mutations through large deletions can be detected with the H P R T test in V79 ceils (Thacker and Ganesh, 1989; Thacker et al., 1990; K6berle and Speit, 1991). By means of cytogenetic and molecular methods we now characterised CBC-induced gene mutations in the in vitro H P R T test in relation to other genetic endpoints. As the induction of chromosome aberrations and lethality by alkylating agents and nitrogen mustard could be enhanced by DNA repair inhibitors like 3-aminobenzamide and caffeine (Lau and Pardee, 1982; Das et al., 1984; Jan et al., 1986), we furthermore studied the effect of inhibited repair on CBC-induced mutations. Materials and methods
The experiments were performed with a clonal derivative of the V79 Chinese hamster cell line.
Ceils were cultivated in minimal essential medium (MEM) with Earle's salts supplemented with 10% foetal calf serum and antibiotics in a humidified incubator at 37°C with 5% CO 2 at a pH of 7.2. Mutagen treatment was performed about 24 h after the start of the experimental subcultures. The test substances used were chlorambucil (CBC), 3-aminobenzamide (3AB) and caffeine (CAF). They were all purchased from Sigma (Munich, Germany). For the H P R T gene mutation test, about 5 × 106 cells were treated in each experiment for 2 h with CBC alone or for 14 h with CBC in the absence or presence of caffeine. The treated cultures were transferred as needed during the expression period (7 days). 6-Thioguanine-resistant colonies were analysed in 5 replicate petri dishes with 2 × 105 cells each. Colonies from independent cultures were isolated for the molecular analysis. Survival (relative plating efficiency) was determined by plating 200 cells into 4 replicate petri dishes at the end of mutagen treatment (PE 1) and at the end of the expression period (PE 2). The methods for D N A isolation, Southern analysis and multiplex polymerase chain reaction (PCR) analysis, were exactly the same as described in a recent paper (K6berle et al., 1991). For Southern analysis, high-molecular-weight DNA was digested with either EcoRI or BgllI and hybridised with a [32p]dCTP nick-translated hprt cDNA probe ( p H P T 12). The same DNA samples were used for PCR reactions. Exons 2-9
TABLE 1 SCE F R E Q U E N C I E S I N D U C E D BY CBC AND T H E I N F L U E N C E OF 3AB AND CAF Treatment
SCEs/cell + SEM
(CBC)
Protocol 1 a
Control 10 -7 M 2 x 10 -7 5)
