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A comparative study of the cytotoxic and genotoxic effects of cisplatin and its analogue, TNO-6, in yeast
Mutation Research, 198 (1988) 69-72
69
Elsevier MTR 04547
A comparative study of the cytotoxic and genotoxic effects of cisplatin and its analogue, TNO-6, in yeast M o h a m m e d A. H a n n a n Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211 (Saudi Arabia)
(Received 1 June 1987) (Revisionreceived 1 September1987) (Accepted14 September1987)
Keywords: Cisplatin; TNO-6; Mitotic gene conversion; (Saccharomyces cerevisiae).
Summary The antitumour drug cisplatin and its analogue, TNO-6, were studied for their cytotoxic, mutagenic and recombinagenic effects in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. It was observed that the structural change in TNO-6 resulted in reduced cytotoxicity and recombinagenicity (mitotic gene conversion) but increased mutagenic activity compared to the effects of cisplatin at equimolar concentrations. These results indicated that the mechanism through which TNO-6 damages cellular DNA is different from that of cisplatin.
Although cisplatin proved to be a clinically active drug against a number of neoplasms (Roberts and Thompson, 1979; Rosenberg, 1980), its toxic side effects warranted a search for new analogues with higher therapeutic index (Bradner et al., 1980; Rose et al., 1982). One of the analogues is TNO-6 which appeared to be less nephrotoxic than cisplatin in rodents (Lee et al., 1983; Lelieveld et al., 1984) and was considered for clinical evaluation. While a large number of studies addressed the possible mechanisms underlying the cytotoxic and genotoxic properties of cisplatin (Anderson, 1979; Roberts and Thompson, 1979; Filipsky et al., 1980; Correspondence: MohammedA. Hannah, Ph.D., Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211 (Saudi Arabia).
Rahn et al., 1980; Zwelling et al., 1981; Hannan et al., 1984), little information is available on the cellular and genetic effects of newly developed analogues. This communication reports data from a comparative study of cytotoxic and genotoxic properties of cisplatin and its analogue, TNO-6. Materials and methods Yeast strain
A wild-type diploid strain of the yeast Saccharomyces cerevisiae designated D7 was used. This strain was kindly supplied by Dr. F.K. Zimmermann. Its genotype has been already described (Zimmermann et al., 1975; Kern and Zimmermarm, 1978). Two non-complementing alleles (trp5-12 and trp5-27) of the gene resulting in tryptophan requirements by the D7 strain enable detection of
0027-5107/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
70
mitotic gene conversion leading to tryptophan independence. As the same strain is also homozygous for the recessive gene, ilvl-92, concerned with a deficiency in isoleucine biosynthesis it allows a study of induced mutations leading to isoleucine independence. Both mitotic gene conversion and mutation were used as the genetic endpoints in the D7 yeast strain to characterize the genotoxic properties of cisplatin and TNO-6.
Media YEPD (yeast extract 1%, bactopeptone 2%, dextrose 2% and agar 2%) was used to grow and maintain yeast cultures. Synthetic complete medium (SC) was adopted from von Borstel et al. (1971). SC lacking tryptophan or isoleucine was used, respectively, to score tryptophan gene convertants and isoleucine non-requiring mutants. Chemicals Cisplatin (cis-diamminodichloroplatinum II) was obtained from Polysciences Inc. while TNO-6 was provided by the Bristol-Myers Company. Both drugs were dissolved and diluted in sterile distilled water in which the yeast cells were treated. The structural features of the two drugs are shown in Fig. 1. Cell treatment and plating procedure Stationary-phase yeast cells were harvested from liquid YEPD by centrifugation and resuspended in sterile distilled water. Cells (at a density of 107/ml) were treated in glass tubes containing water with different concentrations of the drugs for a fixed period of time (2 h). The treated cells were harvested by centrifugation and washed twice and then resuspended in water. Untreated cells prepared the same way served as control.
Appropriate dilutions of the cell suspensions were spread onto petri dishes containing either SC (100-200 cells/plate) or selective medium, i.e., SC lacking tryptophan or isoleucine (106 cells/plate). Cell viability was estimated, by the colony-forming ability of cells on SC while tryptophan gene convertants and isoleucine-independent mutants were scored on the selective medium following 3-6 days of incubation of the plates at 28 ° C. The cytotoxic and genetic effects of the two drugs were compared at equimolar concentrations. Results and discussion
The cell survival curves obtained following treatment with various concentrations of cisplatin and TNO-6 are shown in Fig. 2. It can be seen that, at equimolar concentrations, TNO-6 is much less cytotoxic in the D7 yeast cells than cisplatin.
100
-
~10(Z ul
zl CISPLATIN
N
/cl
o
TNO-6
\Cl CISPLATIN
~
NH2 ~ pt/OSO3
~/
\o~
TNO-6
Fig. 1. Structural features of cisplatin and TNO-6.
1
o
i
I;)
1'5
2'o
~5
3'o
35
DRUG CONCENTRATION (XIO6M)
Fig. 2. Cell survival curves of the D7 yeast strain following treatment with cisplatin (z~) and its analogue, TNO-6 ((3).
71 As cisplatin is known to become more cytotoxic in water by formation of DNA-reactive aquated species (Rahn et al., 1980; Hannan et al., 1984), both drugs (cisplatin and TNO-6) were used in water in order to compare their toxicity. The reduction in the cell killing effects of TNO-6 relative to cisplatin could be due to either reduced solubility/permeability to the yeast cells or altered biological function resulting from the structural difference. It was, thus, necessary to investigate biological effects other than cell killing. Results obtained with cisplatin and TNO-6 on mutagenic and recombinagenic (mitotic gene conversion) activity are shown in Figs. 3 and 4, respectively. It was interesting to find that, at equimolar concentrations, there was an increase in the mutagenic effect of TNO-6 while its recombinagenic activity was remarkably reduced (comparing the frequency per survivor). These results thus reflect a possible relationship between cytotoxicity and
700 -
~
150
>
re
O9
< I~ 100 > z
8 z < z
i ~
°
50-
IN
500 0
5
10 1'8 2'0 2'5 DRUG CONCENTRATION (X 1(~5 M)
3'0
36
Fig. 4. Frequenciesof tryptophangene convertantsinducedby cisplatin (zx)and TNO-6 (©) at equimolarconcentrations.
400
O9 E 0> > n-
IZ I-
> ~ 200 w
!
w co o z
CISP~TIN
o~0-6
100-
0
0
w
0
8
1;) 1'5 2() 2'8 DRUG CONCENTRATION (XIO-$M)
3()
3'8
Fig. 3. Frequenciesof isoleucine-independentmutants induced by cisplatin (zx) and TNO-6 (©) at equimolar concentrations.
DNA lesions which lead to mitotic recombination. Furthermore, the data indicate that the reduction in cytotoxicity and recombinagenicity of TNO-6 is not correlated with its mutagenic potential (frequency/survivor). The results reported here could be interpreted to mean that the DNA lesions responsible for mutations and mitotic recombination or cytotoxicity are different and that the structural change in TNO-6 resulted in the production of more mutagenic lesions and fewer of those responsible for recombinagenic or cytotoxic effects. It is believed that the aquated species (mono and diaquo forms) of cisplatin are most reactive with DNA and that the presence of chloride in the medium reduces its cytotoxic effects by inhibiting the process of aquation (Rahn et al., 1980). Subsequently it has been shown that both genotoxic and cytotoxic effects of cisplatin are reduced in phosphate-buffered saline compared to water (Hannan
72
et al., 1984). It is also believed that cisplatin induces both interstrand and intrastrand crosslinks in DNA and the former mostly accounts for cell death (Filipsky et al., 1980; Zwelling et al., 1981). In an earlier study using DNA repair-deficient mutants of S. cerevisiae, we argued in the favour of two major types of D N A lesions caused by cisplatin; one (interstrand crosslinks) leading to cell death and recombination (through repairmediated generation of strand breaks) and the other (intrastrand crosslinks or base damage) leading to mutations (Hannan et al., 1984). In TNO-6, one of the chloride ligands of cisplatin has been replaced with OH 2 and the other with SO4. While the biological activity of the diamminomethylcyclohexane side of TNO-6 is unknown, it is suggested that the replacement of a chloride ligand with sulphate is responsible for reduced interstrand crosslink formation in D N A and, thence, making it less cytotoxic and recombinagenic. However, a replacement of the other chloride ligand with OH 2 increases its mutagenic property through single-strand damage/base modification compared to that of cisplatin. This possibility can be tested by chemical analysis. This communication only demonstrates a differential effect of TNO-6 on mutagenicity and recombinagenicity, and suggests that both reduced cytotoxicity and recombinagenicity may result from reduced interstrand crosslinks and DNA strand breaks, which, in turn, could be attributed to the structural feature of the drug. If the use of alkaline elution or sucrose gradient techniques confirms these assumptions (reduced interstrand crosslinks and D N A strand breaks by TNO-6), it would be necessary to consider TNO-6 as a drug functionally completely different from cisplatin, and, hence alter the strategy for its combination with other chemotherapeutic agents in order to achieve synergistic effects for cell killing. References Anderson, K.S. (1979) Platinum (II) complexes generate frameshift mutations in test strains of Salmonella typhimurium, Mutation Res., 76, 209-214.
Bradner, W.T., W.C. Rose and J.B. Huftalen (1980) Antitumor activity of platinum analogs, in: A.W. Prestayko, S.T. Crook and S.K. Carter (Eds.), Cisplatin: Current Status and New Developments, Academic Press, New York, Ch. 9, pp. 171-182. Filipsky, J., K.W. Kohn and W.M. Bonner (1980) The nature of inactivating lesions produced by platinum (II) complexes in phage DNA, Chem.-Biol. Interact., 32, 321-330. Hannan, M.A., S.G. Zimmer and J. Hazle (1984) Mechanisms of cisplatin (cis-diammino-dichloroplatinum II) induced cytotoxicity and genotoxicity in yeast, Mutation Res., 127, 23-30. Kern, R., and F.K. Zimmermann (1978) The influence of defects in excision and error prone repair on spontaneous and induced mitotic recombination and mutation in Saccharomyces cerevisiae, Mol. Gen. Genet., 161, 81-88. Lee, F.H., R. Canetta, B.F. Issell and L. Lenaz (1983) New platinum complexes in clinical trials, Cancer Treatm. Rev., 10, 39-51. Lelieveld, P., W.J.F. Van der Vijgh, R.W. Veldhuizen, D. Van Velzen, L.M. Van Putten, G. Atassi and A. Danguy (1984) Preclinical studies on toxicity, antitumor activity and pharmacokinetics of cisplatin and three recently developed derivatives, Eur. J. Cancer Clin. Oncol., 20, 1087-1104. Rahn, R.O., N.P. Johnson, A.W. Hsie, J.F. Lemontt, W.E. Masker, J.D. Regan, W.C. Dunn, J.D. Hoeschele and D.H. Brown (1980) The interaction of platinum compounds with the genome: Correlation between DNA binding and biological effects, in: H.R. Witschi (Ed.), The Scientific Basis of Toxicity, Assessment, Elsevier/North-Holland, Amsterdam, pp. 153-162. Roberts, J.J., and A.J. Thompson (1979) The mechanisms of action of antitumor platinum compounds, in: Progress in Nucleic Acid Research and Molecular Biology, Vol. 22, Academic Press, New York, pp. 71-129. Rose, W.C., J.E. Schurig, J.B. Huftalen and W.T. Bradner (1982) Antitumor activity and toxicity of cisplatin analogs, Cancer Treatm. Rep., 66(1), 135-146. Rosenberg, B. (1980) Cisplatin: Its history and possible mechanisms of action, in: A.W. Prestayko, S.T. Crooke and S.K. Carter (Eds.), Cisplatin: Current Status and New Developments, Academic Press, New York, Ch. 2, pp. 9-19. Von Borstel, R.C., K.T. Cain and C.M. Steinberg (1971) Inheritance of spontaneous mutability in yeast, Genetics, 69, 17-27. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for the simultaneous detection of induced mitotic crossing over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388. Zwelling, L.A., M.O. Bradley, N.A. Sharkey, T. Anderson and K.W. Kohn (1981) DNA crosslinking as an indicator of sensitivity and resistance of mouse L1210 of leukemia to cis-aminodichloroplatinum (II) and L-phenylalanine mustard, Cancer Res., 41,640-649.
69
Elsevier MTR 04547
A comparative study of the cytotoxic and genotoxic effects of cisplatin and its analogue, TNO-6, in yeast M o h a m m e d A. H a n n a n Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211 (Saudi Arabia)
(Received 1 June 1987) (Revisionreceived 1 September1987) (Accepted14 September1987)
Keywords: Cisplatin; TNO-6; Mitotic gene conversion; (Saccharomyces cerevisiae).
Summary The antitumour drug cisplatin and its analogue, TNO-6, were studied for their cytotoxic, mutagenic and recombinagenic effects in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. It was observed that the structural change in TNO-6 resulted in reduced cytotoxicity and recombinagenicity (mitotic gene conversion) but increased mutagenic activity compared to the effects of cisplatin at equimolar concentrations. These results indicated that the mechanism through which TNO-6 damages cellular DNA is different from that of cisplatin.
Although cisplatin proved to be a clinically active drug against a number of neoplasms (Roberts and Thompson, 1979; Rosenberg, 1980), its toxic side effects warranted a search for new analogues with higher therapeutic index (Bradner et al., 1980; Rose et al., 1982). One of the analogues is TNO-6 which appeared to be less nephrotoxic than cisplatin in rodents (Lee et al., 1983; Lelieveld et al., 1984) and was considered for clinical evaluation. While a large number of studies addressed the possible mechanisms underlying the cytotoxic and genotoxic properties of cisplatin (Anderson, 1979; Roberts and Thompson, 1979; Filipsky et al., 1980; Correspondence: MohammedA. Hannah, Ph.D., Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211 (Saudi Arabia).
Rahn et al., 1980; Zwelling et al., 1981; Hannan et al., 1984), little information is available on the cellular and genetic effects of newly developed analogues. This communication reports data from a comparative study of cytotoxic and genotoxic properties of cisplatin and its analogue, TNO-6. Materials and methods Yeast strain
A wild-type diploid strain of the yeast Saccharomyces cerevisiae designated D7 was used. This strain was kindly supplied by Dr. F.K. Zimmermann. Its genotype has been already described (Zimmermann et al., 1975; Kern and Zimmermarm, 1978). Two non-complementing alleles (trp5-12 and trp5-27) of the gene resulting in tryptophan requirements by the D7 strain enable detection of
0027-5107/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
70
mitotic gene conversion leading to tryptophan independence. As the same strain is also homozygous for the recessive gene, ilvl-92, concerned with a deficiency in isoleucine biosynthesis it allows a study of induced mutations leading to isoleucine independence. Both mitotic gene conversion and mutation were used as the genetic endpoints in the D7 yeast strain to characterize the genotoxic properties of cisplatin and TNO-6.
Media YEPD (yeast extract 1%, bactopeptone 2%, dextrose 2% and agar 2%) was used to grow and maintain yeast cultures. Synthetic complete medium (SC) was adopted from von Borstel et al. (1971). SC lacking tryptophan or isoleucine was used, respectively, to score tryptophan gene convertants and isoleucine non-requiring mutants. Chemicals Cisplatin (cis-diamminodichloroplatinum II) was obtained from Polysciences Inc. while TNO-6 was provided by the Bristol-Myers Company. Both drugs were dissolved and diluted in sterile distilled water in which the yeast cells were treated. The structural features of the two drugs are shown in Fig. 1. Cell treatment and plating procedure Stationary-phase yeast cells were harvested from liquid YEPD by centrifugation and resuspended in sterile distilled water. Cells (at a density of 107/ml) were treated in glass tubes containing water with different concentrations of the drugs for a fixed period of time (2 h). The treated cells were harvested by centrifugation and washed twice and then resuspended in water. Untreated cells prepared the same way served as control.
Appropriate dilutions of the cell suspensions were spread onto petri dishes containing either SC (100-200 cells/plate) or selective medium, i.e., SC lacking tryptophan or isoleucine (106 cells/plate). Cell viability was estimated, by the colony-forming ability of cells on SC while tryptophan gene convertants and isoleucine-independent mutants were scored on the selective medium following 3-6 days of incubation of the plates at 28 ° C. The cytotoxic and genetic effects of the two drugs were compared at equimolar concentrations. Results and discussion
The cell survival curves obtained following treatment with various concentrations of cisplatin and TNO-6 are shown in Fig. 2. It can be seen that, at equimolar concentrations, TNO-6 is much less cytotoxic in the D7 yeast cells than cisplatin.
100
-
~10(Z ul
zl CISPLATIN
N
/cl
o
TNO-6
\Cl CISPLATIN
~
NH2 ~ pt/OSO3
~/
\o~
TNO-6
Fig. 1. Structural features of cisplatin and TNO-6.
1
o
i
I;)
1'5
2'o
~5
3'o
35
DRUG CONCENTRATION (XIO6M)
Fig. 2. Cell survival curves of the D7 yeast strain following treatment with cisplatin (z~) and its analogue, TNO-6 ((3).
71 As cisplatin is known to become more cytotoxic in water by formation of DNA-reactive aquated species (Rahn et al., 1980; Hannan et al., 1984), both drugs (cisplatin and TNO-6) were used in water in order to compare their toxicity. The reduction in the cell killing effects of TNO-6 relative to cisplatin could be due to either reduced solubility/permeability to the yeast cells or altered biological function resulting from the structural difference. It was, thus, necessary to investigate biological effects other than cell killing. Results obtained with cisplatin and TNO-6 on mutagenic and recombinagenic (mitotic gene conversion) activity are shown in Figs. 3 and 4, respectively. It was interesting to find that, at equimolar concentrations, there was an increase in the mutagenic effect of TNO-6 while its recombinagenic activity was remarkably reduced (comparing the frequency per survivor). These results thus reflect a possible relationship between cytotoxicity and
700 -
~
150
>
re
O9
< I~ 100 > z
8 z < z
i ~
°
50-
IN
500 0
5
10 1'8 2'0 2'5 DRUG CONCENTRATION (X 1(~5 M)
3'0
36
Fig. 4. Frequenciesof tryptophangene convertantsinducedby cisplatin (zx)and TNO-6 (©) at equimolarconcentrations.
400
O9 E 0> > n-
IZ I-
> ~ 200 w
!
w co o z
CISP~TIN
o~0-6
100-
0
0
w
0
8
1;) 1'5 2() 2'8 DRUG CONCENTRATION (XIO-$M)
3()
3'8
Fig. 3. Frequenciesof isoleucine-independentmutants induced by cisplatin (zx) and TNO-6 (©) at equimolar concentrations.
DNA lesions which lead to mitotic recombination. Furthermore, the data indicate that the reduction in cytotoxicity and recombinagenicity of TNO-6 is not correlated with its mutagenic potential (frequency/survivor). The results reported here could be interpreted to mean that the DNA lesions responsible for mutations and mitotic recombination or cytotoxicity are different and that the structural change in TNO-6 resulted in the production of more mutagenic lesions and fewer of those responsible for recombinagenic or cytotoxic effects. It is believed that the aquated species (mono and diaquo forms) of cisplatin are most reactive with DNA and that the presence of chloride in the medium reduces its cytotoxic effects by inhibiting the process of aquation (Rahn et al., 1980). Subsequently it has been shown that both genotoxic and cytotoxic effects of cisplatin are reduced in phosphate-buffered saline compared to water (Hannan
72
et al., 1984). It is also believed that cisplatin induces both interstrand and intrastrand crosslinks in DNA and the former mostly accounts for cell death (Filipsky et al., 1980; Zwelling et al., 1981). In an earlier study using DNA repair-deficient mutants of S. cerevisiae, we argued in the favour of two major types of D N A lesions caused by cisplatin; one (interstrand crosslinks) leading to cell death and recombination (through repairmediated generation of strand breaks) and the other (intrastrand crosslinks or base damage) leading to mutations (Hannan et al., 1984). In TNO-6, one of the chloride ligands of cisplatin has been replaced with OH 2 and the other with SO4. While the biological activity of the diamminomethylcyclohexane side of TNO-6 is unknown, it is suggested that the replacement of a chloride ligand with sulphate is responsible for reduced interstrand crosslink formation in D N A and, thence, making it less cytotoxic and recombinagenic. However, a replacement of the other chloride ligand with OH 2 increases its mutagenic property through single-strand damage/base modification compared to that of cisplatin. This possibility can be tested by chemical analysis. This communication only demonstrates a differential effect of TNO-6 on mutagenicity and recombinagenicity, and suggests that both reduced cytotoxicity and recombinagenicity may result from reduced interstrand crosslinks and DNA strand breaks, which, in turn, could be attributed to the structural feature of the drug. If the use of alkaline elution or sucrose gradient techniques confirms these assumptions (reduced interstrand crosslinks and D N A strand breaks by TNO-6), it would be necessary to consider TNO-6 as a drug functionally completely different from cisplatin, and, hence alter the strategy for its combination with other chemotherapeutic agents in order to achieve synergistic effects for cell killing. References Anderson, K.S. (1979) Platinum (II) complexes generate frameshift mutations in test strains of Salmonella typhimurium, Mutation Res., 76, 209-214.
Bradner, W.T., W.C. Rose and J.B. Huftalen (1980) Antitumor activity of platinum analogs, in: A.W. Prestayko, S.T. Crook and S.K. Carter (Eds.), Cisplatin: Current Status and New Developments, Academic Press, New York, Ch. 9, pp. 171-182. Filipsky, J., K.W. Kohn and W.M. Bonner (1980) The nature of inactivating lesions produced by platinum (II) complexes in phage DNA, Chem.-Biol. Interact., 32, 321-330. Hannan, M.A., S.G. Zimmer and J. Hazle (1984) Mechanisms of cisplatin (cis-diammino-dichloroplatinum II) induced cytotoxicity and genotoxicity in yeast, Mutation Res., 127, 23-30. Kern, R., and F.K. Zimmermann (1978) The influence of defects in excision and error prone repair on spontaneous and induced mitotic recombination and mutation in Saccharomyces cerevisiae, Mol. Gen. Genet., 161, 81-88. Lee, F.H., R. Canetta, B.F. Issell and L. Lenaz (1983) New platinum complexes in clinical trials, Cancer Treatm. Rev., 10, 39-51. Lelieveld, P., W.J.F. Van der Vijgh, R.W. Veldhuizen, D. Van Velzen, L.M. Van Putten, G. Atassi and A. Danguy (1984) Preclinical studies on toxicity, antitumor activity and pharmacokinetics of cisplatin and three recently developed derivatives, Eur. J. Cancer Clin. Oncol., 20, 1087-1104. Rahn, R.O., N.P. Johnson, A.W. Hsie, J.F. Lemontt, W.E. Masker, J.D. Regan, W.C. Dunn, J.D. Hoeschele and D.H. Brown (1980) The interaction of platinum compounds with the genome: Correlation between DNA binding and biological effects, in: H.R. Witschi (Ed.), The Scientific Basis of Toxicity, Assessment, Elsevier/North-Holland, Amsterdam, pp. 153-162. Roberts, J.J., and A.J. Thompson (1979) The mechanisms of action of antitumor platinum compounds, in: Progress in Nucleic Acid Research and Molecular Biology, Vol. 22, Academic Press, New York, pp. 71-129. Rose, W.C., J.E. Schurig, J.B. Huftalen and W.T. Bradner (1982) Antitumor activity and toxicity of cisplatin analogs, Cancer Treatm. Rep., 66(1), 135-146. Rosenberg, B. (1980) Cisplatin: Its history and possible mechanisms of action, in: A.W. Prestayko, S.T. Crooke and S.K. Carter (Eds.), Cisplatin: Current Status and New Developments, Academic Press, New York, Ch. 2, pp. 9-19. Von Borstel, R.C., K.T. Cain and C.M. Steinberg (1971) Inheritance of spontaneous mutability in yeast, Genetics, 69, 17-27. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for the simultaneous detection of induced mitotic crossing over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388. Zwelling, L.A., M.O. Bradley, N.A. Sharkey, T. Anderson and K.W. Kohn (1981) DNA crosslinking as an indicator of sensitivity and resistance of mouse L1210 of leukemia to cis-aminodichloroplatinum (II) and L-phenylalanine mustard, Cancer Res., 41,640-649.