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Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes
Environmental Mutagenesis
ELSEVIER
Mutation Research 334 (1995) 19-22
Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes D. Mourelatos
a,* A.
Papageorgiou
b L.
Boutis c, p. Catsoulacos d
a Department of General Biology and Genetics, Faculty of Medicine, Aristotelian University, Thessaloniki 54006, Greece b Department of Chemotherapy, Theagenion Cancer Institute, Thessaloniki, Greece c Department of Pharmacology, Faculty of Medicine, Aristotelian University, Thessaloniki, Greece d Department of Pharmacy, Laboratory of Pharmaceutical Chemistry, University of Patras, Greece Received 11 January 1994; revision received 24 March 1994; accepted 29 March 1994
Abstract
The effect of P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3[3-hydroxy-N-methyl-17a-aza-D-homo-5aandrostan-17-one (compound 3) and 3/3-hydroxy-17a-aza-o-homo-5a-androstane (compound 2) on sister-chromatid exchange (SCE) frequencies and on human lymphocytes proliferation kinetics was studied. The results are compared with those of the P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3fl-hydroxy-17a-aza-D-homo-5c~-androstan17-one (compound 1). All compounds were found to be active in inducing markedly increased SCE rates and cell division delays. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumour activity of these compounds was observed.
Keywords." Homo-aza-steroidal esters; Sister-chromatid exchange; Cell kinetics 1. Introduction
The homo-aza-steroidal esters of carboxylic derivatives of the nitrogen mustard N,N-bis(2chloroethyl)aniline display antitumour activity (Catsoulacos, 1984; Catsoulacos et al., 1988). The presence of the - N H - C O - group in the steroidal molecule of the A or D ring affects the antitumour activity since the c2¢tostatic results are superior to those obtained with unmodified steroidal esters (Wall et al., 1969). This in combination with the fact that compound 1 inhibits DNA synthesis (Papageorgiou et al., 1983) prompted us
* Corresponding author. Elsevier Science B.V.
SSDI 0 1 6 5 - 1 1 6 1 ( 9 4 ) 0 0 0 2 7 - M
to study the extent and the nature of the contribution of the lactamic group to the cytogenetic and antitumour activity. For this purpose compounds 2 and 3 were recently synthesised (Figs. 2 and 3). In both cases modifications have been effected in the - N H - C O - group of compound 1, which is a very active representative of this series of homo-aza-steroidal esters containing alkylating agents. In the first case the hydrogen of the N H group has been substituted with methyl (Fig. 3), and in the other case the - N H - C O - group has been reduced to - N H C H 2- (Fig. 2). A comparative study, on a molar basis, of the three compounds as regards their ability to induce SCEs and cell division delays was undertaken and the results were correlated with the previously established antitumour activity of these
20
D. Mourelatos et al. / Mutation Research 334 (1995) 19-22
agents. SCEs in vitro (Tofilon et al., 1985) and in vivo (Mourelatos et al., 1988; Petrou et al., 1990; Lialiaris et al., 1992) have been proposed as one of the methods for evaluating chemotherapeutic efficiency.
Table 1 Induction of SCEs and cell division delays by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenylacetate in h u m a n lymphocytes Agent and concentration (/x M) Control 1
2. Materials and methods
Lymphocyte cultures were prepared by adding eight drops of heparinsed whole blood from two normal subjects to 5 ml of chromosome medium (Gibco 1A). The cultures were incubated for 96 h at 37°C. Metaphases were collected during the last 2 h with coichicine at 0 . 3 / x g / m l . Treatment with the chemicals, which were dissolved in the same chromosome medium, was given 18 h after initiation of culture. For SCE demonstration 5bromodeoxyuridine (BrdUrd) at 4 /zg/ml was given 24 h after initiation of culture. Throughout all cultures were maintained in the dark to minimise photolysis of BrdUrd. Air-dried preparations were made and stained with the F P G procedure (Goto et al., 1978). The preparations were scored for cells in their first mitosis (both chromatids darkly stained), second mitosis (one chromatid of each chromosome darkly stained) and third and subsequent divisions (a portion of chromosomes with both chromatids lightly stained) and suitably spread second-division cells were scored for SCEs. A minimum of 30 cells was scored for each culture. For proliferation rate indices (PRIs) 100 cells at least were scored. SCEs and PRIs were scored in coded slides. For the statistical evaluation of the experimental data the chi-square test was used for the cell kinetic comparisons, whereas for the SCE frequencies Student's t-test was performed to determine whether any values deviated significantly from the controls ( P < 0.05). We also calculated the correlation between the PRIs and the SCE frequencies.
3. Results and discussion
Results are presented in Table 1 and chemical formulae of the drugs used in Figs. 1-3. All three
SCE/celI+ SEa
PRI
7.10 _+0.49
1.93
Compound l 0.2 0.4 0.6
45.50_+ 1.91 61.86_+2.18 80.18 _+2.22
1.82 1.50 1.32
Compound 2 0.2 0.4 0.6
20.39 + 0.82 41.64+ 1.16 55.55 _+ 1.30
1.79 1.43 1.34
Compound 3 0.2 0.4 0.6
34.60 +_ 1.51 48.66+_ 1.19 57.88 _+2.33
1.56 1.60 1.33
6.15 + 0.28
1.86
Compound l 0.2 0.4 0.6
29.54 _+0.76 39.15 +_0.82 50.68 _+ 1.16
1.70 1.59 1.37
Compound 2 0.2 0.4 0.6
26.03 +_ 1.04 35.07_+ 1.56 43.30_+ 1.94
1.67 1.53 1.34
Compound 3 0.2 0.4 0.6
17.73 +_0.74 26.97 _+0.79 36.06 _+ 1.67
1.73 1.60 1.37
Control 2
a For establishing mean SCE value 30-40 second-division cells were counted for each point. PRIs were calculated as (1M1 + 2 M 2 + 3 M 3 + ) / 1 0 0 , where M1 is the percent value of cells in the first, M2 in the second and M 3 + in the third higher divisions. The PRIs have been correlated with corresponding SCE values (r = - 0 . 7 , P < 0.001).
compounds induced marked increases in SCE frequencies and cell division delays at all concentrations tested (0.2, 0.4 and 0.6 /xM). The increases were directly related to the concentration tested. Compound 1 was the most effective SCEinducing agent with compounds 2 and 3 following. The cell division delays were directly related to the doses used. The results in Table 1 seem to show that a correlation in the group of three
21
D. Mourelatos et al. / Mutation Research 334 (1995) 19-22
14
CH3 I
CICH2CH2\ /~ ~t .H~2 C O O " / " CI CH2CH7~~. -. - tC
Fig. 1. 3fl-Hydroxy-13c~-amino-13,17-seco-5a-androstan-17 oic- 13,17-1actam-p-N, N-bis(2-chloroethyl)aminophenylacetate (compound 1).
substances exists: the higher the PRI value the lower the SCE frequency. Most antitumour agents have been identified to be either clastogenic, carcinogenic, mutagenic or teratogenic (Seiber and Adamson, 1975; Au et al., 1980). Chemically induced cytotoxicity, in that it delays cell turnover times, is clearly manifested as a change in the relative proportions of cells in their first, second and subsequent divisions (Morimoto, 1983). Studies in search of a relationship between SCE induction and other expressions of genotoxicity have shown a positive relationship between SCEs and reduced cell survival and alteration in cell cycle kinetics (Morris and Heflich, 1984). All three compounds (Fig. 1-3) were previously simultaneously tested for anticancer activity in vivo against leukaemia L1210, P388, Lewis lung carcinoma and Ehrlich ascites tumour. These compounds contain a modified steroid as a biological platform for transporting the alkylating agent to the tumour site. The antitumour activity of compound 1 was distinctly superior to that of compounds 2 and 3 (Catsoulacos et al., 1992) in all four experimental animal tumours and leukaemias examined in vivo. The cytogenetic
H
CI
H2COO
H
Fig. 2. 3O-Hydroxy-17a-aza-D-homo-5a-androstan-p-N,Nbis(2-chloroethyl)aminophenylacetate (compound 2).
[//%~/.~//~..,0 v H ~ v
Fig. 3. 3/3-Hydroxy-N-methyJ-]Ta-aza-D-homo-5a-androstan-
p-N,N-bis(2-chlorocthyl)aminophenyJacetate(compound 3).
(Table 1) and antineoplastic (Catsoulacos et al., 1992) effects might be better in compound 1 than in compounds 2 and 3 due to the possibility of multiple interactions of the - N H C O - lactam group (Fig. 1) with similar groups which exist in DNA and proteins and the possibility that the lactam nucleus is transformed by a metabolic process or at least by an enzymatically catalyzed reaction to the active species: -NHCO-
~- N - H e -C=O *
Compounds 2 and 3, which come next in order of effectiveness in SCE induction, do not contain the - N H C O - group but they have either the =N-H or the =C-O group (Figs. 2 and 3) which may account for the cytogenetic and antineoplastic effects achieved by these two compounds. An attempt was made to correlate the known differential antitumour activity of these compounds with their effectiveness in SCE induction. There are findings indicating that the effectiveness in SCE induction by antitumour alkylating agents in rodent tumour cells in vitro and in vivo can be positively correlated with the in vivo tumour response to these agents, and these findings suggest that the SCE assay could be used to predict both the sensitivity of human tumour cells to chemotherapeutic agents and the heterogeneity of drug sensitivity within individual tumours (Lialiaris et al., 1992; Deen et al., 1986). The SCE assay has predictive value as a clinical assay for drugs for which a strong correlation between cell killing and induction of SCEs has been established (Deen et al., 1986). In the present work, for the three chemicals studied, a sound correlation between SCE induction and cell division
22
D. Mourelatos et al. /Mutation Research 334 (1995) 19-22
delay (P < 0.001, Table 1) and previously established (Catsoulacos et al., 1992) antitumour activity was observed.
References Au, W. D.A. Johnston, A. Collie-Bruy~re and T.C. Hsu (1980) Short term cytogenetic assays of 9 cancer chemotherapeutic drugs with metabolic activation, Environ. Mutagen., 2, 455-464. Catsoulacos, P. (1984) Further studies on the antineoplastic activity of 3/3-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic- 13,17-1actam-p-bis(2-chloroethyl)aminophenylacetate, Cancer Lett., 22, 199-202. Catsoulacos, P. D. Politis and G.L. Wampler (1988) Antitumour activity of homo-aza-steroidal esters of p-bis(2chloroethyl)aminophenylacetic acid and p-bis(2-chloroethyl)aminophenylbutyric acid, Cancer Chemother. Pharmacol., 7, 129-132. Catsoulacos, P. C. Camoutsis, M. Pelecanou, A. Papageorgiou and E. Adamiak-Margariti (1992) Cytostatic effects of homo-aza-steroidal esters in vivo and in vitro, Structureactivity relationships, Anticancer Res., 12, 1617-1620. Deen, D.F., L.A. Kendall, L.J. Marton and P.J. Tofilon (1986) Prediction of human tumour cell chemosensitivity using the SCE assay, Cancer Res., 46, 1599-1602. Goto, K., S. Maeda, Y. Kano and T. Sugiyama (1978) Factors involved in the differential Giemsa-staining of sister chromatids, Chromosoma, 66, 351-359. Lialiaris, t., A. Pantazaki, E. Sivridis and D. Mourelatos (1992) Chlorpromazine induced damage on nucleic acids:
a combined cytogenetic and biochemical study, Mutation Res., 265, 155-163. Morimoto, K. (1983) Induction of SCEs and cell division delays in human lymphocytes by microsomal activation of benzene, Cancer Res., 43, 1330-1334. Morris, S.M. and R.H. Heflich (1984) A comparison of the toxic and SCE-inducing effects of inhibitors of ADP-ribosyltransferase in CHO cells, Mutation Res., 126, 63-71. Mourelatos, D. J. Dozi-Vassiliades, A. Kotsis and C. Gourtsas (1988) Enhancement of cytogenetic damage and of antineoplastic effect by caffeine in Ehrlich ascites tumour cells treated with cyclophosphamide in vivo, Cancer Res., 48, 1129-1131. Papageorgiou, A. G. Wanov, S.I. Koliais, L. Boutis and P. Catsoulacos (1983) Interaction of homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenylacetic acid with DNA of Ehrlich ascites tumour cells, FEBS lett., 153, 194-198. Petrou, C. D. Mourelatos, E. Mioglou, J. Dozi-Vassiliades and P. Catsoulacos (1990) Effects of alkylating antineoplastics alone or in combination with 3-aminobenzamide on genotoxicity, antitumour activity and NAD levels n human lymphocytes in vitro and on Ehrlich ascites tumour cells in vivo, Teratogen. Carcinogen Mutagen, 10, 321-331. Seiber, S.M. and R.H. Adamson (1975) The clastogenic, mutagenic, teratogenic and carcinogenic effects of various antineoplastic agents, in: Pharmacologic Basis of Cancer Chemotherapy, M.D. Anderson Hospital and Tumour Institute of Houston. Williams and Wilkins, Baltimore, MD, pp 401-468. Tofilon, P.J.I. Basic and L. Milas (1985) Prediction of in vivo tumour response to chemotherapeutic agents by the in vitro SCE assay, Cancer Res., 45, 225-2030. Wall, M.E., G.S. Abernethy, F.G. Carrol and D.J. Taylor (1969) The effect of some steroidal alkylating agents on experimental animal mammary tumor and leukemia systems, J. Med. Chem., 12, 810-818.
ELSEVIER
Mutation Research 334 (1995) 19-22
Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes D. Mourelatos
a,* A.
Papageorgiou
b L.
Boutis c, p. Catsoulacos d
a Department of General Biology and Genetics, Faculty of Medicine, Aristotelian University, Thessaloniki 54006, Greece b Department of Chemotherapy, Theagenion Cancer Institute, Thessaloniki, Greece c Department of Pharmacology, Faculty of Medicine, Aristotelian University, Thessaloniki, Greece d Department of Pharmacy, Laboratory of Pharmaceutical Chemistry, University of Patras, Greece Received 11 January 1994; revision received 24 March 1994; accepted 29 March 1994
Abstract
The effect of P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3[3-hydroxy-N-methyl-17a-aza-D-homo-5aandrostan-17-one (compound 3) and 3/3-hydroxy-17a-aza-o-homo-5a-androstane (compound 2) on sister-chromatid exchange (SCE) frequencies and on human lymphocytes proliferation kinetics was studied. The results are compared with those of the P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3fl-hydroxy-17a-aza-D-homo-5c~-androstan17-one (compound 1). All compounds were found to be active in inducing markedly increased SCE rates and cell division delays. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumour activity of these compounds was observed.
Keywords." Homo-aza-steroidal esters; Sister-chromatid exchange; Cell kinetics 1. Introduction
The homo-aza-steroidal esters of carboxylic derivatives of the nitrogen mustard N,N-bis(2chloroethyl)aniline display antitumour activity (Catsoulacos, 1984; Catsoulacos et al., 1988). The presence of the - N H - C O - group in the steroidal molecule of the A or D ring affects the antitumour activity since the c2¢tostatic results are superior to those obtained with unmodified steroidal esters (Wall et al., 1969). This in combination with the fact that compound 1 inhibits DNA synthesis (Papageorgiou et al., 1983) prompted us
* Corresponding author. Elsevier Science B.V.
SSDI 0 1 6 5 - 1 1 6 1 ( 9 4 ) 0 0 0 2 7 - M
to study the extent and the nature of the contribution of the lactamic group to the cytogenetic and antitumour activity. For this purpose compounds 2 and 3 were recently synthesised (Figs. 2 and 3). In both cases modifications have been effected in the - N H - C O - group of compound 1, which is a very active representative of this series of homo-aza-steroidal esters containing alkylating agents. In the first case the hydrogen of the N H group has been substituted with methyl (Fig. 3), and in the other case the - N H - C O - group has been reduced to - N H C H 2- (Fig. 2). A comparative study, on a molar basis, of the three compounds as regards their ability to induce SCEs and cell division delays was undertaken and the results were correlated with the previously established antitumour activity of these
20
D. Mourelatos et al. / Mutation Research 334 (1995) 19-22
agents. SCEs in vitro (Tofilon et al., 1985) and in vivo (Mourelatos et al., 1988; Petrou et al., 1990; Lialiaris et al., 1992) have been proposed as one of the methods for evaluating chemotherapeutic efficiency.
Table 1 Induction of SCEs and cell division delays by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenylacetate in h u m a n lymphocytes Agent and concentration (/x M) Control 1
2. Materials and methods
Lymphocyte cultures were prepared by adding eight drops of heparinsed whole blood from two normal subjects to 5 ml of chromosome medium (Gibco 1A). The cultures were incubated for 96 h at 37°C. Metaphases were collected during the last 2 h with coichicine at 0 . 3 / x g / m l . Treatment with the chemicals, which were dissolved in the same chromosome medium, was given 18 h after initiation of culture. For SCE demonstration 5bromodeoxyuridine (BrdUrd) at 4 /zg/ml was given 24 h after initiation of culture. Throughout all cultures were maintained in the dark to minimise photolysis of BrdUrd. Air-dried preparations were made and stained with the F P G procedure (Goto et al., 1978). The preparations were scored for cells in their first mitosis (both chromatids darkly stained), second mitosis (one chromatid of each chromosome darkly stained) and third and subsequent divisions (a portion of chromosomes with both chromatids lightly stained) and suitably spread second-division cells were scored for SCEs. A minimum of 30 cells was scored for each culture. For proliferation rate indices (PRIs) 100 cells at least were scored. SCEs and PRIs were scored in coded slides. For the statistical evaluation of the experimental data the chi-square test was used for the cell kinetic comparisons, whereas for the SCE frequencies Student's t-test was performed to determine whether any values deviated significantly from the controls ( P < 0.05). We also calculated the correlation between the PRIs and the SCE frequencies.
3. Results and discussion
Results are presented in Table 1 and chemical formulae of the drugs used in Figs. 1-3. All three
SCE/celI+ SEa
PRI
7.10 _+0.49
1.93
Compound l 0.2 0.4 0.6
45.50_+ 1.91 61.86_+2.18 80.18 _+2.22
1.82 1.50 1.32
Compound 2 0.2 0.4 0.6
20.39 + 0.82 41.64+ 1.16 55.55 _+ 1.30
1.79 1.43 1.34
Compound 3 0.2 0.4 0.6
34.60 +_ 1.51 48.66+_ 1.19 57.88 _+2.33
1.56 1.60 1.33
6.15 + 0.28
1.86
Compound l 0.2 0.4 0.6
29.54 _+0.76 39.15 +_0.82 50.68 _+ 1.16
1.70 1.59 1.37
Compound 2 0.2 0.4 0.6
26.03 +_ 1.04 35.07_+ 1.56 43.30_+ 1.94
1.67 1.53 1.34
Compound 3 0.2 0.4 0.6
17.73 +_0.74 26.97 _+0.79 36.06 _+ 1.67
1.73 1.60 1.37
Control 2
a For establishing mean SCE value 30-40 second-division cells were counted for each point. PRIs were calculated as (1M1 + 2 M 2 + 3 M 3 + ) / 1 0 0 , where M1 is the percent value of cells in the first, M2 in the second and M 3 + in the third higher divisions. The PRIs have been correlated with corresponding SCE values (r = - 0 . 7 , P < 0.001).
compounds induced marked increases in SCE frequencies and cell division delays at all concentrations tested (0.2, 0.4 and 0.6 /xM). The increases were directly related to the concentration tested. Compound 1 was the most effective SCEinducing agent with compounds 2 and 3 following. The cell division delays were directly related to the doses used. The results in Table 1 seem to show that a correlation in the group of three
21
D. Mourelatos et al. / Mutation Research 334 (1995) 19-22
14
CH3 I
CICH2CH2\ /~ ~t .H~2 C O O " / " CI CH2CH7~~. -. - tC
Fig. 1. 3fl-Hydroxy-13c~-amino-13,17-seco-5a-androstan-17 oic- 13,17-1actam-p-N, N-bis(2-chloroethyl)aminophenylacetate (compound 1).
substances exists: the higher the PRI value the lower the SCE frequency. Most antitumour agents have been identified to be either clastogenic, carcinogenic, mutagenic or teratogenic (Seiber and Adamson, 1975; Au et al., 1980). Chemically induced cytotoxicity, in that it delays cell turnover times, is clearly manifested as a change in the relative proportions of cells in their first, second and subsequent divisions (Morimoto, 1983). Studies in search of a relationship between SCE induction and other expressions of genotoxicity have shown a positive relationship between SCEs and reduced cell survival and alteration in cell cycle kinetics (Morris and Heflich, 1984). All three compounds (Fig. 1-3) were previously simultaneously tested for anticancer activity in vivo against leukaemia L1210, P388, Lewis lung carcinoma and Ehrlich ascites tumour. These compounds contain a modified steroid as a biological platform for transporting the alkylating agent to the tumour site. The antitumour activity of compound 1 was distinctly superior to that of compounds 2 and 3 (Catsoulacos et al., 1992) in all four experimental animal tumours and leukaemias examined in vivo. The cytogenetic
H
CI
H2COO
H
Fig. 2. 3O-Hydroxy-17a-aza-D-homo-5a-androstan-p-N,Nbis(2-chloroethyl)aminophenylacetate (compound 2).
[//%~/.~//~..,0 v H ~ v
Fig. 3. 3/3-Hydroxy-N-methyJ-]Ta-aza-D-homo-5a-androstan-
p-N,N-bis(2-chlorocthyl)aminophenyJacetate(compound 3).
(Table 1) and antineoplastic (Catsoulacos et al., 1992) effects might be better in compound 1 than in compounds 2 and 3 due to the possibility of multiple interactions of the - N H C O - lactam group (Fig. 1) with similar groups which exist in DNA and proteins and the possibility that the lactam nucleus is transformed by a metabolic process or at least by an enzymatically catalyzed reaction to the active species: -NHCO-
~- N - H e -C=O *
Compounds 2 and 3, which come next in order of effectiveness in SCE induction, do not contain the - N H C O - group but they have either the =N-H or the =C-O group (Figs. 2 and 3) which may account for the cytogenetic and antineoplastic effects achieved by these two compounds. An attempt was made to correlate the known differential antitumour activity of these compounds with their effectiveness in SCE induction. There are findings indicating that the effectiveness in SCE induction by antitumour alkylating agents in rodent tumour cells in vitro and in vivo can be positively correlated with the in vivo tumour response to these agents, and these findings suggest that the SCE assay could be used to predict both the sensitivity of human tumour cells to chemotherapeutic agents and the heterogeneity of drug sensitivity within individual tumours (Lialiaris et al., 1992; Deen et al., 1986). The SCE assay has predictive value as a clinical assay for drugs for which a strong correlation between cell killing and induction of SCEs has been established (Deen et al., 1986). In the present work, for the three chemicals studied, a sound correlation between SCE induction and cell division
22
D. Mourelatos et al. /Mutation Research 334 (1995) 19-22
delay (P < 0.001, Table 1) and previously established (Catsoulacos et al., 1992) antitumour activity was observed.
References Au, W. D.A. Johnston, A. Collie-Bruy~re and T.C. Hsu (1980) Short term cytogenetic assays of 9 cancer chemotherapeutic drugs with metabolic activation, Environ. Mutagen., 2, 455-464. Catsoulacos, P. (1984) Further studies on the antineoplastic activity of 3/3-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic- 13,17-1actam-p-bis(2-chloroethyl)aminophenylacetate, Cancer Lett., 22, 199-202. Catsoulacos, P. D. Politis and G.L. Wampler (1988) Antitumour activity of homo-aza-steroidal esters of p-bis(2chloroethyl)aminophenylacetic acid and p-bis(2-chloroethyl)aminophenylbutyric acid, Cancer Chemother. Pharmacol., 7, 129-132. Catsoulacos, P. C. Camoutsis, M. Pelecanou, A. Papageorgiou and E. Adamiak-Margariti (1992) Cytostatic effects of homo-aza-steroidal esters in vivo and in vitro, Structureactivity relationships, Anticancer Res., 12, 1617-1620. Deen, D.F., L.A. Kendall, L.J. Marton and P.J. Tofilon (1986) Prediction of human tumour cell chemosensitivity using the SCE assay, Cancer Res., 46, 1599-1602. Goto, K., S. Maeda, Y. Kano and T. Sugiyama (1978) Factors involved in the differential Giemsa-staining of sister chromatids, Chromosoma, 66, 351-359. Lialiaris, t., A. Pantazaki, E. Sivridis and D. Mourelatos (1992) Chlorpromazine induced damage on nucleic acids:
a combined cytogenetic and biochemical study, Mutation Res., 265, 155-163. Morimoto, K. (1983) Induction of SCEs and cell division delays in human lymphocytes by microsomal activation of benzene, Cancer Res., 43, 1330-1334. Morris, S.M. and R.H. Heflich (1984) A comparison of the toxic and SCE-inducing effects of inhibitors of ADP-ribosyltransferase in CHO cells, Mutation Res., 126, 63-71. Mourelatos, D. J. Dozi-Vassiliades, A. Kotsis and C. Gourtsas (1988) Enhancement of cytogenetic damage and of antineoplastic effect by caffeine in Ehrlich ascites tumour cells treated with cyclophosphamide in vivo, Cancer Res., 48, 1129-1131. Papageorgiou, A. G. Wanov, S.I. Koliais, L. Boutis and P. Catsoulacos (1983) Interaction of homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenylacetic acid with DNA of Ehrlich ascites tumour cells, FEBS lett., 153, 194-198. Petrou, C. D. Mourelatos, E. Mioglou, J. Dozi-Vassiliades and P. Catsoulacos (1990) Effects of alkylating antineoplastics alone or in combination with 3-aminobenzamide on genotoxicity, antitumour activity and NAD levels n human lymphocytes in vitro and on Ehrlich ascites tumour cells in vivo, Teratogen. Carcinogen Mutagen, 10, 321-331. Seiber, S.M. and R.H. Adamson (1975) The clastogenic, mutagenic, teratogenic and carcinogenic effects of various antineoplastic agents, in: Pharmacologic Basis of Cancer Chemotherapy, M.D. Anderson Hospital and Tumour Institute of Houston. Williams and Wilkins, Baltimore, MD, pp 401-468. Tofilon, P.J.I. Basic and L. Milas (1985) Prediction of in vivo tumour response to chemotherapeutic agents by the in vitro SCE assay, Cancer Res., 45, 225-2030. Wall, M.E., G.S. Abernethy, F.G. Carrol and D.J. Taylor (1969) The effect of some steroidal alkylating agents on experimental animal mammary tumor and leukemia systems, J. Med. Chem., 12, 810-818.