Toxicity, interstrand cross-links and DNA fragmentation induced by ‘activated’ cyclophosphamide in yeast: Comparative studies on 4-hydroperoxy-cyclophosphamide, its monofunctional analogon, acrolein, phosphoramide mustard, and nor-nitrogen mustard

Chem.-Biol. Interactions, 39 (1982) 1--15

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Elsevier/North-HollandScientific Publishers Ltd.

TOXICITY, INTERSTRAND CROSS-LINKS AND DNA FRAGMENTATION INDUCED BY 'ACTIVATED' CYCLOPHOSPHAMIDE IN YEAST: COMPARATIVE STUDIES O N 4-HYDROPEROXY-CYCLOPHOSPHAMIDE, ITS MONOFUNCTIONAL ANALOGON, ACROLEIN, PHOSPHORAMIDE MUSTARD, AND NOR-NITROGEN MUSTARD

R. FLEER and M. BRENDEL*

lnstitut fiir Mikrobioiogie, Universit~t Frankfurt, Theodor-Stern-Kai 7, Haus 75, 6000 Frankfurt/M (F.R. G.)

(Received December 27th, 1980) (Revision received July 20th, 1981) (Accepted September 14th, 1981) SUMMARY Activated cyclophosphamide (CP) is known to achieve its cytotoxic and alkylating capacity upon spontaneous hydrolytic breakdown of the oxazaphosphorine ring structure. Treatment of yeast cells with the chemically activated form of CP (4-hydroperoxy-CP, 4-OOH-CP) and with several potentiatly toxic cleavage products revealed that cytotoxicity is closely linked to the formation of DNA interstrand cross-links and to DNA fragmentation. While this holds true for 4-OOH-CP and its bifunctional alkylating breakdown products, phosphoramide mustard (PM) and nor-nitrogen mustard (NNM), equimolar concentrations of acrolein and the monofunctional analogon of activated CP were inactive. NNM, the ultimate cleavage product within the successive degradation of the oxazaphosphorine structure was five times more toxic than 4-OOH-CP, whereas the cytotoxic action of PM was only slightiy enhanced. The high cytotoxicity of NNM was matched by its ability to induce DNA interstrand cross-links: at concentrations and treatment times producing equal cell killing, 4-OOH-CP and NNM produced the same extent of cross-linking and DNA fragmentation. Biochemical potency of NNM is in contrast to data found with the NBP colorimetric assay which suggest that NNM loses its alkylating activity at neutral pH. 4-OOH-CP and PM are much more stable than predicted from half-life measurements performed via the NBP colorimetric assay: they retain a considerable fraction of their cytotoxic and cross-linking activity in spite of a 12-h preincubation at pH 7 and 36°C. *To whom correspondenceshould be sent. Abbreviations: CP, cyclophosphamide; 5t-dTMP, deoxythymidine-5t-monophosphate; NNM, nor-nitrogen mustard; 4-OOH-CP, 4-hydroperoxy-CP;PM, phosphoramide mustard; TCA, trichloroacetic acid. 0009--2797/82/0000--0000/$02.75 © Elsevier/North-HollandScientificPublishers Ltd.

INTRODUCTION Because o f its broad spectrum of activity against human and animal tumors, CP is one of the most extensively used agents in the chemotherapy of cancer. In order to elucidate its molecular mechanism of cytotoxicity and its action on DNA within the living cell, we recently proposed a simple in vitro test system which makes use of the unicellular eucaryotic organism S. cerevisiae [1--3]. With a chromatin structure closely similar to that of higher developed organisms [4], yeast proves to be well suited for the analysis of DNA alterations and allows to by-pass the specific problems of experimentation with mammalian cells. With this test system we could demonstrate induction of DNA interstrand cross-links and DNA fragmentation in parallel to cell toxicity and mutagenesis after CP treatment of repair deficient haploid yeast [2,3]. Using a method that allows detection of, in the average, less than one cross-link per yeast chromosome, this effect could already be demonstrated at the lowest concentration that exerted a measurable cytotoxic effect in an alkylation sensitive strain. Results were essentially the same whether the CP treatment was performed with the chemically activated form of CP in vitro (4~)OH-CP) or whether we treated yeast with CP in vivo employing the host-mediated assay [3]. Activated CP does neither exhibit cytotoxic nor alkylating properties until 'toxification' occurs by cleavage of the oxazaphosphorine ring system [5]: hydrolytic breakdown results in the release of the toxic aldehyde acrolein and the bifunctionally alkylating PM. Furthermore, the alkylating agent NNM has been detected in blood and urine of patients treated with CP [6--8]. Finally, a number of metabolites without alkylating activity as well as monofunctionaUy alkylating cleavage products of CP will be formed in vivo [9,10]. So far considerable controversy exists about the molecular mechanism of CP action as well as the metabolites ultimately responsible for the therapeutic and toxic effects of this drug [10]. In this study, we therefore investigated the action of mono
Strain, media and conditions o f growth In all experiments but one the haploid, excision deficient strain MB10722B was used. It is thermoconditionally auxotrophic for deoxythymidine-5'monophosphate (5'-dTMP) and a highly efficient 5'
A lkylating agents 4-OOH-CP, mono
of Dr. G. Peter, Gustav-Embden-Zentrum der Biologischen Chemie, Frank-

furt/M. Treatment o f cells and measurement o f inactivation, renaturability and D N A

fragmentation All procedures have already been described in detail [2,11]. Briefly,treatments with alkylatingagents have been carried out with radioactivelylabeled, stationary haploid yeast cellssuspended in phosphate buffer (0.067 M; p H 7) at 36°C. After stopping the reaction by several washings, ceilswere either resuspended in buffer for postincubation experiments or immediately frozen in an Eaton press at -70°C. Homogenization was performed in a crushing mixture containing 2 5 % glycerol. The assay of D N A interstrand cross-links is based on the technique of reversibledenaturation: covalent linkage of the D N A sister strands will allow renaturation of alkali
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Fig. 1. Influence of a 12oh preincubation of 4-OOH-CP on its cytotoxic activity; • •, 10 -4 M 4-OOH-CP, no preincubation; o------o, cells treated with 4-OOH-CP preincubated in 0.067 M phosphate buffer (pH 7; 36°C) for 12 h, initial concentration 10 -4 M; 0-------o, same procedure as before, preincubation, however, was performed in presence of 4 x 10 ~ yeast cells/ml;-, 1.5x 10 -s M 4-OOH-CP, no preincubation. corresponding to the remaining activity of the preincubated solution when calculating tl/2 to 4.2 h [5], we find the decrease in c y t o t o x i c activity caused b y t h e preincubation to be much smaller than expected. While we do not observe any inactivation of cells after a 12-h treatment with 1.5 X 10 -s M 4-OOH-CP, there is about 85% cell killing after 12 h whether w e use preincubated or freshly dissolved 10 -4 M 4-OOH-CP. A marked loss of cytotoxicity caused b y preincubation of the c o m p o u n d is only seen at longer treatment, though still smaller than expected. When 10 -4 M 4~)OHCP is preincubated in the presence of 4 X 107 yeast cells/ml and cells are removed after 12 h, loss o f c y t o t o x i c activity of t h e supernatant is more pronounced than that caused b y the preincubation procedure in buffer alone (Fig. 1). This indicates binding o f some active agent to t h e cells. As t h e persistence o f c y t o t o x i c activity must n o t reflect the stability of bifunctional alkylating activity, we investigated possible differences in t h e level o f DNA primary lesions induced b y 4-OOH-CP with or without pre-

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incubation. The amount of renaturable DNA (a measure of interstrand cross-linking) as well as the sedimentation in sucrose gradients (a measure of DNA fragmentation) after a 12-h treatment of cells with both preincubated and freshly dissolved 4-OOH-CP are depicted in Fig. 2: treatment leading to about the same inhibition of cell multiplication does induce the same renaturability of DNA, the same number of double- and single~trand breaks and thus the same number of DNA interstrand cross-links. These findings indicate that either: (i) PM, the proposed alkylating metabolite of 4-OOH-CP, is much more stable than expected from the NBP data, (ii) NNM (tl/2 = 6.3 hi5]) and not PM is responsible for the toxicity of pre. incubated solutions of 4-OOH-CP or (iii) the cytotoxic action of 4 ~ ) O H ~ P is mediated by stable cleavage products such as acrolein or some monoo functional breakdown products of activated CP. The role of the aldehyde acrolein which is released from 4~)OH-CP in equimolar concentrations as PM is still discussed controversially: whereas it is generally thought to be much less toxic than the parent compound [9,10], a considerable contribution to cell killing and chromosome damage has been reported by some authors [14--16]. Therefore, we investigated possible biological and biochemical effects of this metabolite. However, at least in the dose range employed in our 4-OOH-CP experiments, acrolein does neither attribute to the cytotoxic effects o f 4-OOH-CP (Fig. 3) nor induce interstrand cross-links or DNA fragmentation (Fig. 4) in yeast. To ensure that toxicity and DNA damage are not a consequence of an unspecific action of partially hydrolyzed 4-OOH-CP that would lead to monofunctionaUy alkylating break
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Fig. 2. Influence of preincubation of 4-OOH-CP on its alkylating activity. Cells were treated for 12 h, solutions of 10 -4 M 4-OOH-CP were either freshly prepared or preincubated in 0.067 M phosphate buffer (pH 7; 36°C) for 12 h. (a) Renaturability of DNA as measured in neutral CsCI equilibrium density gradients: o o, preincubation; 0 - - - - - - % no preincubation (untreated control similar to Fig. 4a; right and left bands define positions of renatured and denatured DNA, respectively). (b) Fragmentation of DNA as determined by sedj= mentation of native (double-stranded) DNA in neutral, calibrated 15--30% sucrose gradients: v u, preincubation; ~ , - - - z~, no premcubation; • 8, untreated control. (c) Sedimentation of denatured (single-stranded) DNA in neutral 15--30% sucrose gradients, o v and ~ - - - - - - % preincubation; o - - - - - - o , no preincubation; • J, untreated control. ArroWs indicate respective molecular weights.

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Fig. 3. Lethal e f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s o f N N M , 4-OOH-CP, m o n o - d e c h l o r o - 4 OOH-CP and PM o n yeast as a f u n c t i o n o f t r e a t m e n t time. Left panel: • =, 10 -3 M N N M ; v - - - - - - - v , s o l u t i o n o f N N M preincubated in 0 . 0 6 7 M p h o s p h a t e buffer (pH 7; 36°C) for 14 h, initial c o n c e n t r a t i o n 10 -3 M; • • , 10 -4 M N N M ; • A, 2 X 1 0 -3 M NNM; n--.---~, 1 0 - ' M 4-OOH-CP; o o, 10 -4 M m o n o - d e c h l o r o - 4 - O O H - C P ; O - - - - - ©, 10 -4 M acrolein. Right panel: • =, 10 -3 M P M ; • •, 10 -4 M P M ; • A, 2 X 10-3 M pM; ~ - - • ---~, 10-4 M 4.OOH.CP.

that the half-lifedata obtained with the N B P assay are also relevant for the decay of biological activity. "' To test this hypothesis we investigated the stability of the cytotoxic activity of P M in analogy to the preincubation experiments performed with 4-OOH-CP. The decay of toxicity at different times of preincubation is shown in Fig. 5b: although further experiments are necessary to determine the precise haLE-lifetime (a rough estimation yields a ti/2 of 6 h) our results clearly show that solutions of P M retain a considerable fraction of their cytotoxic activity even after 14 h of preincubation: there is still7 5 % of cell killing, whereas a hydrolytic decay of PM, equivalent to about 3 half-life times, would lead to 100% survival under the conditions of the test (Fig. 5a). At short preincubation times, we even observe an increase of toxicity which indicates the formation of a metabolite either more reactive with, or taken up more readily by the cell. In order to discriminate between toxic effects due to mechanisms other than bifunctional alkylation, analogous experiments were performed with a mutant selectively sensitive against bi_functional alkylating agents. The

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Fig. 4. Influence of mono-dechloro-4-OOH-CP and acrolein o n renaturability and molecular weight of DNA. Cells were treated with 10 -4 M mono-dechloro-4-OOH-CP, 10 -4 M acrolein or equimolar 4-OOH-CP for 12 h. (a) Banding of alkali denatured and reneutralizad DNA in isopycnie CsCI gradients: v - - . - - - o , mono-dechloro-4-OOH-CP; o - - - - - - o , acrolein; • •, 4-OOH-CP; • A, untreated control. (b) Sedimentation of denatured (single-stranded) DNA in neutral, calibrated 15--30% sucrose gradients; symbols equivalent to Fig. 4a.

mutation is thermoconditional, with treated cells sensitive at 36°C but with wild type resistance at 23°C [19]. At both temperatures, the mutant is resistant (i.e. wild type) against monofunctionally alkylating agents. Treatment of this strain with preincubated solutions of PM reveals that there is a higher toxicity at 36°C as compared to incubation at 23°C (Fig. 5). The difference in the sensitivity at 23°C and 36°C decreases only slightly upon further preincubation of the PM solution which is an indirect measure for the prolonged integrity of both chloroethyl residues. A rapid hydrolysis of the first side chain would result in a corresponding convergency of the two inactivation curves.

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Fig. 5. Stability of biological activity o f PM. (a) Inactivation kinetics of strain F51 after treatment with PM at a concentration of 10 -4 M ( o - - - - - v ) and 10 -s M (o v). The arrow indicates the treatment time applied in the p~eincobation experiments. (b) Decay of cytotoxicity of PM as a function of preineubation time. In general, cells were treated for 2 h at 36°C. o o, initial concentration of PM was 10-* M, experiments performed with strain F51; o a, initial concentration was 10 .4 M. Ceils (strain F51) were treated for 11 h, however, e - - - - - l , inactivation of the e n m l ts m u t a n t (initial conc. of PM -- 10-* M), colonies grown at 23°C; = - - . - i , inactivation of the immg ts mutant (initial cone. of PM = 10 -s M), colonies grown at 36°C.

To confirm the persistence o f bifunctionally aikylating activity during the preincubation of PM and NNM in a more direct approach, we followed the D N A cross-linking ability o f both compounds: PM as well as NNM were able to induce D N A inters/zand croM-lin~s in yeast; NNM, however, proved to be more active. This finding agrees well with the higher toxicity o f NNM ( d . Fig. 3; Table I). Solutions o f PM as well as o f NNM retain a marked

10 TABLE I COMPARING THE DNA CROSS-LINKING POTENCY OF NNM AND PM Yeast cells were treated at 10-3 M with both compounds for 2 h and at 36°C. % Renaturable DNAa

NNM PM Control

No preincubation

12-h preincubation

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a Further increase of renaturable DNA during postincubation of treated cells (cf. Ref. 11) was not considered; data are not corrected for DNA strand breakage. f r a c t i o n o f t h e i r b i f u n c t i o n a l a l k y l a t i n g activity u p o n p r e i n c u b a t i o n f o r 1 2 - - 1 4 h (Table I). T h e r e f o r e we c a n n o t d e c i d e t o w h a t e x t e n t cross-linking caused b y p r e i n c u b a t e d PM is due t o t h e c o m p o u n d p r o p e r o r t o its cleavage p r o d u c t NNM. Since N N M is t h e u l t i m a t e b r e a k < l o w n p r o d u c t w i t h i n t h e successive d e c a y o f activated CP and since it p r o v e d t o b e m o r e t o x i c t h a n 4-OOH-CP and PM, ~ e c o m p a r e d its effects o n D N A w i t h t h o s e o f e q u i t o x i c c o n c e n t r a -

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Fig. 7. Influence o f postincubation o f cells on renaturability of DNA: effects of equitoxic concentrations o f NNM and 4-OOH-CP. Sedimentation in neutral CsCI equilibrium density gradients. (a) [] v, cells treated with 2 X 10 -s M NNM for 7.3 h, survival 38%; o - - - - - - o , 10 -4 M 4-OOH-CP, survival 40%. (b) [] a, 2 X 10 -s M NNM for 7.3 h and additional 3 h of postincubation in 0.067 M phosphate buffer (pH 7; 36°C); o - - - - - o , 10 -4 M 4-OOH-CP for 8 h and 4 h o f postincubation. (c) [] a, 2 X 10 -5 M NNM for 7.3 h and 22 h o f postincubation; o-- -- --o, 10 -4 M 4-OOH-CP for 8 h and 20 h of postincubation (control values similar to Fig. 4a). 107

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Fig. 8. Influence of postincubation of cells on molecular weight of DNA: effects o f equitoxic concentrations o f NNM and 4-OOH-CT. Sedimentation o f native (doublestranded) DNA in neutral, calibrated 15~30% sucrose gradients, o--o - - o cells treated with 2 x 10 -s M NNM for 7.3 h, postincubation was 22 h; o - - - - - - c , 10 -4 M 4-OOH-CP for 8 h, postincubation was 20 h; • ; , untreated control, incubation in buffer for 24 h.

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tions of 4-OOH-CP: at the same level of cell killing, we found the same extent of DNA interstrand cross-linking u p o n direct treatment as well as after postincubation of cells (Figs. 6 and 7). Also, we observed comparable extents of DNA fragmentation after action of both compounds (Fig. 8). DISCUSSION

In contrast to what would be expected from appropriate NBP measurements [5], the alkylating activity of 4-OOH-CP proved to be stable for a long period of time under physiological conditions of temperature and pH. Cell killing after a 12-h treatment with 4-OOH-CP is alike, irrelevant of a preincubation of this compound for 12 h which should be equivalent to about 3 half-life times according to the NBP data (Fig. 1). At shorter treatment times, inactivation of cells seems even more pronounced when using preincubated solutions of 4-OOH-CP as compared to the freshly dissolved c o m p o u n d (Fig. 1). This may be explained as follows: since 4~)OH-CP gains its toxicity only after release of non-cyclic alkylating compounds [5], toxication will already have happened to a certain extent after preincubation of 4-OOH-CP. Therefore cell killing will already be observed at short treatment times. On the other hand, detoxication of active cleavage products by hydrolysis of the chloroethyl moiety will occur at the same time. Thus, the total exposure concentration of alkylating metabolites will be r e t u c e d on preincubation, resulting in a less steep slope of the inactivation curve (Fig. 1). Since at equal cell killing we find an equal extent of interstrand crosslinking and DNA fragmentation (Fig. 2) whether preincubated or freshly dissolved 4-OOH-CP is applied, the bifunctionally alkylating activity must be refractive to hydrolytic breakdown over a number of hours. This assumption is supported by the fact that neither acrolein nor the monofunctional analogon to 4-OOH-CP do contribute to the toxic and DNA damaging effects of the parent compound (Figs. 3 and 4). Cytotoxicity proves to be closely linked to the appearance of DNA interstrand cross-links and DNA strand breakage. Both types of DNA lesions are known to possess considerable lethal potential [20,21]. In procaryotic organisms a single interstrand cross-link has been shown to correspond to one lethal hit, provided that DNA repair is prevented [22,23]. Cross-linking as well as DNA degradation can be achieved by treatment of cells with 4~OOH-CP, PM and NNM. More detailed studies of the kinetics of biological and biochemical effects of 4-OOH-CP towards yeast in vitro and in the host mediated assay have been reported recently [2,3]. Cross-link induction b y PM has also been demonstrated in mouse leukemia cells by means of the alkaline elution assay [24]. Possibly due to differences in cellular uptake, the authors find PM 4 - 5 times less toxic than 4-sulfido-CP derivatives (which, in analogy to 4-OOH-CP, spontaneously generate 4-OH-CP in aqueous solutions). However, the same kinetics of cross-link inductior~ is observed when equltoxic concentrations of PM and the 4~ulfido-CP derivatives are compared. This again indicates a close relation between the amount of cell killing and the extent of DNA interstrand cross-linking. PM is thought to be

13 the ultimate alkylating metabolite responsible for the cytotoxic activity of activated CP [24]. Our data, however, strongly suggest that a further breakdown product of PM, namely NNM, will contribute to toxicity and cross-linking potency of 4-OOH-CP. NNM, which has been shown to be formed to a considerable extent in vitro [12,13] and in vivo [6--8], proved to be the most active compound both in cell killing and induction of DNA damage when compared to 4-OOH-CP and PM. At equal treatment times and at equitoxic concentrations, we find the same extent of interstrand cross-linking after t~eatment of yeast with either NNM or 4~)OH-CP (Fig. 6) as well as after postincubation of cells following withdrawal of excessive agent (Fig. 7). The same holds true when we tested for DNA fragmentation (Fig. 8). Similarly, NNM has been shown to cause chromosome breakage and reduction of mitotic events in CHO cells at about the same" rate as activated CP [16]. These findings are in contrast to what has been postulated from measurements with the NBP assay [13]: there only PM but not NNM behaves as a bifunctionally alkylating agent at neutral pH. The demonstration of DNA interstrand cross-links, induced by NNM at pH 7 (Figs. 6 and 7; Table I), as well as the discrepancy between half-life data obtained with the NBP test on the one hand and the persistence of crosslinking activity of preincubated 4-OOH-CP and PM (Fig. 2; Table I) on the other hand, raises questions about the biological relevance of the colorimetric test system. A different reactivity of alkylating metabolites against defined chemical compounds in vitro and against DNA within the living cell seems likely. At least with respect to the action of NNM, the intracellular pH may play an important role. Many years ago, it has been claimed (see Ref. 9) that NNM, which might be released within the cell, could contribute to the cancerospecific action of CP: the increased alkylating activity of this compound at lower pH could account for a higher cytotoxicity to cancer cells which, due to a higher rate of glycolysis, are known to be more acidic than normal tissues. Further experimentation is necessary to establish the relative role of PM and NNM in the biological activity of activated CP. On the DNA level, use of 32P-labeled compounds should allow direct measurement of the amount of diguaninyl-reaction products exhibiting the phosphoryl group of PM. ACKNOWLEDGEMENTS

This investigation was supported by a grant of the Deutsche Forschungsgemeinschaft. We would like to thank Dr. G. Peter for kindly supplying 4-OOH-CP, mono-dechloro-4-OOH-CP, PM and NNM. We appreciate the skilful drawing of figures by Ms. M. Markovic. This work is part o f the Ph. D. Thesis of the first author. REFERENCES 1 R. Fleer and M. Brendel, Yeast as a model for studying DNA-alterations and biological response induced by alkylating anti-cancer drugs: effects of cyclophospha-

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