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Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1
Mutation Research 373 Ž1997. 215–223
Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1 J.G. Hengstler a
a,)
, A. Hengst a , J. Fuchs a , B. Tanner b, J. Pohl c , F. Oesch
a
Institute of Toxicology, UniÕersity of Mainz, Obere Zahlbacher Str. 67, 55131 Mainz, Germany b Department of Gynecology, UniÕersity of Mainz, 55131 Mainz, Germany c ASTA Medica AG, 60001 Frankfurt, Germany Received 13 June 1996; revised 15 August 1996; accepted 16 August 1996
Abstract Cyclophosphamide requires metabolic activation by cytochrome P450 to exert its genotoxic effects. Therefore in vitro studies on its mechanism of action have been limited to the use of self-activating derivatives of cyclophosphamide or to hepatocytes as an activating system. In this study we used a cell line of Chinese hamster lung fibroblasts ŽV79 cells., genetically engineered to express active cytochrome P450 2B1 as the sole observable cytochrome P450 ŽSD1 cells.. An increase in DNA strand lesions ŽSL: DNA single-strand breaks and alkali labile sites. was observed between 0.5 and 1.5 mM cyclophosphamide Ž24 h incubation. which could be classified as alkali labile sites using a modified alkaline elution assay. Compared to cyclophosphamide, its active metabolite 4-hydroperoxycyclophosphamide Ž4-OOH-CY. was about 250-fold more effective in induction of SL. Equimolar concentrations of phosphoramide mustard Ž50 mM., the ultimate DNA binding metabolite of cyclophosphamide, caused only about 50% of SL compared to 4-OOH-CY. A minimum of 12 h of incubation of SD1 cells was needed for cyclophosphamide Ž1 mM. until SL were detectable, compared to only 2 h for 4-OOH-CY and 1.5 h for phosphoramide mustard Ž50 mM.. DNA crosslinks were observable after shorter incubation periods than single-strand breaks Ž6 h for cyclophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard. and were no longer detectable at incubation periods of more than 20 h. Treatment of SD1 cells with ionizing radiation only, cyclophosphamide only, and radiation plus cyclophosphamide showed that SL induced by cyclophosphamide were not repaired during incubation with fresh culture medium Ž24 h.. However, an efficient repair of SL caused by ionizing radiation was observed and was not inhibited by cyclophosphamide. These observations give strong evidence that different types of SL were induced by cyclophosphamide and radiation. SD1 cells were able to repair the special kind of SL induced by radiation but not the SL caused by cyclophosphamide. Keywords: 4-Hydroxycyclophosphamide; Phosphoramide mustard; Cytochrome P450 2B1; DNA-repair; Alkali labile site; Ionizing radiation
1. Introduction Abbreviations: SL, DNA strand lesion ŽDNA single-strand breaks and alkali labile sites.; 4-OOH-CY, 4-hydroperoxycyclophosphamide ) Corresponding author. Tel.: q49 6131r173115; Fax: q49 6131r230506.
Cyclophosphamide is one of the most often applied antitumor agents w1,2x in clinical practice. However, studies on the mechanism of action of cyclophosphamide at a cellular level have been diffi-
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cult, because cyclophosphamide requires activation by hepatic enzymes ŽFig. 1.. Cyclophosphamide is activated by mixed-function oxidases w3,4x to 4-hydroxycyclophosphamide which spontaneously decomposes to acrolein and phosphoramide mustard. Thus, the requirement for cytochrome P450-mediated activation of cyclophosphamide has limited in vitro studies to the use of self-activating derivatives of cyclophosphamide, such as 4-hydroperoxycyclophosphamide or sulfidocyclophosphamide derivatives w5x or the use of hepatocytes as activating systems w6x. In this study we used SD1 cells, which are Chinese hamster lung fibroblasts ŽV79 cells. genetically engineered to express active cytochrome P450 2B1
as the sole detectable cytochrome P450 w4x. SD1 cells are able to activate cyclophosphamide and due to this they were used in this study to examine the induction and repair of DNA single-strand breaks as well as crosslinks induced by cyclophosphamide and its metabolites phosphoramide mustard and 4-hydroperoxycyclophosphamide Žwhich releases 4-hydroxycyclophosphamide in aqueous solution.. Additionally DNA damaging effects of cyclophosphamide in SD1 cells could be compared to those in mock transfected V79 cells, the parental cell line which does not express any detectable activity of cytochrome P450. Thus, it was possible to differentiate between those effects of cyclophosphamide which can be exerted only after activation by cytochrome P450 2B1 and those which can be induced by cyclophosphamide itself without any activation by cytochrome P450.
2. Materials and methods 2.1. Alkaline elution
Fig. 1. Metabolism of cyclophosphamide.
The alkaline elution method of Kohn et al. w7x was employed with some modifications. A suspension of 2 million cells in 1 ml of cold phosphate-buffered saline ŽPBS. was poured onto polycarbonate filters ŽNucleopore, Tubingen, Germany; 25 mm diameter, ¨ 2 mm pore size.. Cells were lysed with 3 ml of a solution of 10 mM ethylenediaminetetraacetic acid ŽEDTA., 0.5% Triton X-100, 20 M NaCl Žadjusted to pH 10 with NaOH. for 60 min. After the lysis cells were washed with 4.5 ml 10 mM EDTA Žadjusted to pH 10 with NaOH. using a flow rate of 3 mlrh for 90 min. Before starting sampling of the eluted DNA, one fraction equal to the volume of the tube Ž0.25 ml. was discarded. The elution was performed at 48C in the dark using a 5 M NaCl, 20 mM EDTA solution adjusted to pH 12.6 with NaOH. Using a pump speed of 1.5 mlrh, the eluting solution was collected over a period of 10 hrs in a single fraction. After removal of the filters, they were sonicated in 15 ml elution buffer for 2 = 15 min Žfilter fraction.. Quantification of the eluted DNA and of the DNA in the filter fractions was performed using an automated detection system Žcontinuous
J.G. Hengstler et al.r Mutation Research 373 (1997) 215–223
flow detection system; Skalar Analytic, Erkelenz, Germany. which was based on the following treatment: the sample was neutralized with 0.1 M KH 2 PO4 adjusted with K 2 HPO4 Ž0.1 M. to pH 6.0. Thereafter the neutralized solution was mixed with a fluorochrome solution Ž0.1 M K 2 HPO4 adjusted with KH 2 PO4 Ž0.1 M. to pH 7.2; 0.88 mgrl bisbenzimide; Riedel-de-Haen, Seelze, Germany.. The fluorescent complex was measured at an emission wavelength of 450 nm and an excitation wavelength of 365 nm. The elution rate was calculated as yŽlog 10 R . r 10 h, where R represents the fraction of DNA remaining on the filter after 10 h of elution. The described alkaline elution technique was standardized with gamma-irradiation Žcaesium source; 500 cGyrmin., which induced a linear, dose-dependent increase in DNA single-strand breaks. An increase in elution rate of 12 = 10y3 hy1 was induced by 1 Gy. Each sample was eluted on four separate filters and mean values were calculated. For the analysis of total DNA crosslinks, cells were irradiated with 800 cGy prior to elution using a cesium gamma-irradiation source Ždose rate: 500 cGyrmin.. Cells were kept on ice 15 min before and during irradiation. Sensitivity of DNA crosslink analysis was not improved if higher or lower radiation doses were used. For the analysis of the kinetics of formation of alkali labile sites, the solution eluting through the filter was collected in 12 fractions of 60 min each. Elution rates obtained by an elution solution adjusted to pH 12.6 were compared to elution rates obtained by an elution solution adjusted to pH 12.2, as the rate of conversion of alkali labile sites to DNA single-strand breaks is known to be increased with increasing pH w10x. Thus, lower DNA elution rates are observed at pH 12.2 compared to pH 12.6 if alkali labile sites are present, whereas no difference will be observed if only direct DNA single-strand breaks are present.
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SD1 cells were treated with geneticin ŽG 418; 400 mgrml. once a month to select cells transfected with cytochrome P450 2B1 w4x. However, cytotoxicity of cyclophosphamide to SD1 cells was never increased after selection with geneticin during the period of this study, which shows a relatively stable expression of cytochrome P450 2B1. Cells were split in a 1 : 2 ratio 24 h before they were incubated with drugs. Cyclophosphamide, 4-OOH-CY, and phosphoramide mustard were dissolved in Dulbecco’s minimal essential medium and added to half- confluent SD1 cells in 60-mm Petri dishes. Before analysis by alkaline elution the cells were washed with 10 ml of phosphate-buffered saline, harvested from the plate after trypsinization, and the viability was measured by trypan blue exclusion. Phosphoramide mustard and 4-OOH-CY were stored at y208C and added to the culture medium of the cells within 3 min when they had been dissolved.
4. Results A dose-dependent induction of DNA strand lesions ŽSL. by cyclophosphamide and its metabolites 4-hydroxycyclophosphamide and phosphoramide mustard was observed in SD1 cells after 24 h of incubation ŽFigs. 2 and 3.. For cyclophosphamide a significant increase in SL was observed at concentrations between 0.5 and 1.5 mM ŽFig. 2.. No further increase in SL could be observed between 1.5 and 4 mM. In the dose range from 0.5–1.5 mM no statistically significant decrease in trypan blue exclusion was observed compared to control cells ŽFig. 2.. At
3. Cell culture and drug treatment SD1 cells w4x were cultured at 378C, PŽCO. 2 s 5%, with Dulbecco’s minimal essential medium supplemented with 10% foetal calf serum 125 mgrliter of streptomycin, and penicillin Ž0.125 mega unitsrliter..
Fig. 2. Induction of DNA strand lesions Želution rate. and trypan blue exclusion of SD1 cells after incubation with cyclophosphamide for 24 h. Mean values and standard deviations of three independent experiments are given.
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Fig. 3. Induction of DNA strand lesions Želution rate. in SD1 cells after incubation with 4-hydroxycyclophosphamide and phosphoramide mustard for 24 h. Mean values and standard deviations of three independent experiments are given.
concentrations higher than 4 mM a further increase in SL appeared and in the same dose range the SD1 cells lost their ability to exclude trypan blue. Compared to cyclophospham ide, 4-hydroper oxycyclophosphamide Žwhich spontaneously releases 4-hydroxycyclophosphamide . induced similar Fig. 5. Induction of DNA crosslinks Žwhich correlate with the decrease in elution rate. in SD1 cells after incubation with 1 mM hydro peroxcyclophosphamide Ža. and with 50 mM 4-hydroxycyclophosphamide or 50 mM phosphoramide mustard Žb. for various incubation periods. Mean values and standard deviations of three independent experiments are given.
Fig. 4. Induction of DNA strand lesions Želution rate. in SD1 cells after incubation with 2 mM cyclophosphamide Ža. and with 50 mM 4-hydroperoxycyclophosphamide or 50 mM phosphoramide mustard Žb. for various incubation periods. Mean values and standard deviations of three independent experiments are given.
amounts of SL if about 250-fold lower concentrations were used ŽFig. 3.. Equimolar concentrations of phosphoramide mustard caused only about 50% of SL compared to 4-OOH-CY. Up to the highest concentrations of 4-OOH-CY and phosphoramide mustard no statistically significant decrease in trypan blue exclusion was observed. A time-dependent induction of SL was observed after incubation of SD1 cells with 2 mM cyclophosphamide or 50 mM 4-OOH-CY or 50 mM phosphoramide mustard ŽFig. 4.. For cyclophosphamide a statistically significant increase in SL was observed after 12 h of incubation, but not for shorter incubation periods ŽFig. 4a.. However, 4-OOH-CY and phosphoramide mustard induced detectable amounts of SL already after 2 h of incubation ŽFig. 4b.. In order to investigate DNA crosslinks, cells were irradiated with 800 cGy after incubation with cyclophosphamide. DNA crosslinks were detectable after shorter incubation periods than SL ŽFig. 5.. For
J.G. Hengstler et al.r Mutation Research 373 (1997) 215–223
Fig. 6. Alkaline elution profiles of SD1 cells after incubation with 2 mM cyclophosphamide for 24 h and of control cells Ža., as well as SD1 cells after irradiation with 800 cGy and control cells Žb. using elution solutions adjusted to pH 12.2 and pH 12.6. The ordinate gives the logarithm of the percentage of DNA retained on the filters. The abscissa gives the time DNA was eluted through the filter.
cyclophosphamide Ž1 mM. an induction of DNA crosslinks Žwhich was shown by a decrease in elution rates. was observed after 6 h of incubation and reached a maximum after about 15 h. However, no induction of DNA crosslinks was observed if shorter incubation periods were used ŽFig. 5a.. Phosphoramide mustard Ž50 mM. and 4-OOH-CY Ž50 mM. induced detectable amounts of DNA crosslinks already after 1 h of incubation ŽFig. 5b.. A higher level of DNA crosslinks was induced by 4-OOH-CY compared to phosphoramide mustard. To differentiate between direct DNA single-strand breaks and alkali labile sites ŽDNA-lesions which are converted to DNA single-strand breaks by alkaline pH; the rate of conversion increases with increasing
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pH w10x. SD1 cells were incubated with cyclophosphamide Ž2 mM; 48 h. and alkaline elution profiles were compared using an elution solution adjusted to pH 12.6 and 12.2 ŽFig. 6.. SD1 cells irradiated with 800 cGy were used as a control, because ionizing radiation is known to induce predominantly direct DNA single-strand breaks. For the irradiated SD1 cells, no statistically significant difference could be observed using elution solutions adjusted to pH 12.6 and 12.2 ŽFig. 6b.. However, for SD1 cells incubated with cyclophosphamide an about 7-fold higher elution rate was detected at pH 12.6 compared to pH 12.2 ŽFig. 6a.. As the elution of the DNA from control cells was almost identical at pH 12.6 and 12.2, only the data obtained at pH 12.6 are shown in Fig. 6a and b. To examine whether SL induced by cyclophosphamide can be repaired in SD1 cells, cyclophosphamide treated SD1 cells Ž2 mM; 24 h. were washed twice with PBS, the medium containing cyclophosphamide was replaced by fresh medium Žwithout cyclophosphamide. and the cells were further incubated at 378C. No repair of SL was observed up to 3 h of postincubation ŽFig. 7.. The lack in repair of cyclophosphamide-induced SL could be due to an inhibition of DNA repair enzymes by cyclophosphamide. Alternatively SD1 cells could be constitutionally deficient in repair of SL. Therefore we examined DNA repair in SD1 cells which were only treated
Fig. 7. DNA strand lesions Želution rate. in SD1 cells after incubation with 2 mM cyclophosphamide for 24 h and subsequent incubation in fresh medium for 0.5, 1.5, and 3 h. Mean values and standard deviations of three independent experiments are given.
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riod in fresh medium Žwithout cyclophosphamide. the level of SL decreased by the amount of SL which had been induced by ratiation, but did not decline significantly below the level of SL which were induced by treatment with cyclophosphamide only.
5. Discussion
Fig. 8. DNA strand lesions in SD1 cells directly after irradiation with 600 cGy and after subsequent incubation at 378C for 30 min and 3 h Ža. and in SD1 cells after incubation with cyclophosphamide Ž2 mM; 48 h., after incubation with cyclophosphamide Ž2 mM; 48 h. and additional irradiation with 600 cGy, and after a subsequent incubation period in fresh medium without cyclophosphamide Ž378C. for 30 min and 3 h Žb.. Mean values and standard deviations of three independent experiments are given.
with gamma-radiation ŽFig. 8a. and with gammaradiation plus cyclophosphamide ŽFig. 8b.. SD1 cells only treated with 600 cGy gamma-radiation repaired about 60% of the initially induced SL after 30 min postincubation at 378C. After 3 h of incubation the level of SL had declined to the level of the controls ŽFig. 8.a.. Thus, SD1 cells could efficiently repair SL induced by ionizing radiation contrary to those induced by cyclophosphamide. Treatment of SD1 cells with 2 mM cyclophosphamide for 48 h induced an increase of SL to 340% of the control cells ŽFig. 8b.. If SD1 cells were incubated for 48 h with 2 mM cyclophosphamide and additionally irradiated with 600 cGy the elution rate increased to 780% of the control cells. During the subsequent incubation pe-
SD1 cells, which express cytochrome P450 2B1, were able to activate cyclophosphamide w4x. Thus, these cells can be used to investigate the mechanisms of cyclophosphamide toxicity and mutagenicity in the same cells in which the active metabolites are formed and do not require hepatocytes as an external activating system. Cyclophosphamide induced a dose-dependent increase in DNA strand lesions ŽSL. in SD1 cells in a dose range from 0.5–1.5 mM cyclophosphamide. From 1.5-4 mM the dose-effect curve showed no further increase, which may be due to a saturation of cytochrome P450 2B1. Up to 4 mM cyclophosphamide SD1 cells excluded trypan blue. On account of that fact, SL observed up to this concentration resulted from a direct attack to the DNA and not from indirect effects due to a loss of cell membrane integrity w8x. At concentrations higher than 4 mM SD1 cells lost their ability to exclude trypan blue. Thus, SL induced in this concentration range seem to be caused by an indirect effect due to the loss of cell membrane integrity and the release of DNA degrading enzymes from lysosomes w8x. Induction of SL by cyclophosphamide was compared to that of 4-hydroperoxycyclophosphamide Ž4OOH-CY. and phosphoramide mustard. 4-OOH-CY is transported into the cells and spontaneously releases 4-hydroxycyclophosphamide w9,14,15x. In vivo 4-hydroxycyclophosphamide is formed in the liver and transported to the tumor via the blood stream w6x. Phosphoramide mustard is considered to be the ultimate DNA binding metabolite w6x. In the present study 4-OOH-CY was about 2-fold more active in inducing SL in SD1 cells than phosphoramide mustard. The smaller effect of phosphoramide mustard is probably due to the higher polarity of phosphoramide mustard at physiological pH, which renders penetration through the cell membrane more difficult
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w5x or due to the absence of the effect of acrolein, also inducing SL w6x. 4-OOH-CY was about 250-fold more active than cyclophosphamide. Thus, only a relatively small fraction of cyclophosphamide seems to be activated to 4-OOH-CY by SD1 cells. A minimum of 12 h of incubation of SD1 cells was needed for cyclophosphamide Ž2 mM. until SL were detectable, compared to only 2 h for 4-OOH-CY Ž50 mM. and 1.5 h for phosphoramide mustard Ž50 mM.. These differences show that a relatively long period of time was needed by SD1 cells to produce an amount of genotoxic cyclophosphamide metabolites, which were sufficient to induce a detectable level of SL. On the other hand formation of phosphoramide mustard from 4-OOH-CY must be a relatively fast process. DNA strand lesions cause an increased elution of DNA through the filters during alkaline elution, whereas DNA crosslinks decrease DNA elution. On principle, DNA crosslinks should be measurable by the detection of the decreased elution of DNA from control cells through filters. However, at least 85% of the DNA from control cells is retained on the filter using the standard alkaline elution procedure. Thus, the small range from 85 to 100% retention of DNA does not allow a sensitive detection of DNA crosslinks. Owing to that fact, artificial SL are induced by ionizing radiation prior to elution to increase the sensitivity of DNA crosslink detection w10x. We found 600-800 cGy to be an optimal dose range. Using substantially higher doses the sensitivity of crosslink detection decreased again, because DNA fragments became so small that nearly all DNA eluted through the filter, irrespective of whether DNA crosslinks were present or not. In this study DNA crosslinks were detectable after shorter incubation time than SL Ž6 h for cyclophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard.. They were no longer detectable at incubation times longer than 20 h, in contrast to SL which increased up to the longest incubation times tested Ž52 h.. The observed increase in elution rates after a temporary decrease ŽFig. 5. must not be interpreted as repair of DNA crosslinks, because it occurred in the same period, in which an increase in SL was observed ŽFig. 4.. An explanation might be a conversion of crosslinks to alkali labile sites caused by a loss of purine bases as a consequence of destabilization of
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N-glycosidic bonds by bivalent adducts. This is supported by high-performance liquid chromatography analysis of DNA adducts caused by cyclophosphamide, which have shown that cyclophosphamide was able to induce crosslinks between N7 positions of guanine w11x. Those adducts depurinated readily and after 24 h at pH 7.0 and 378C 70% of them had been liberated. This agrees with the present examination, which has classified cyclophosphamide induced SL as alkali labile. Alkali lability has been shown to be typical for depurination sites w10x. The loss of a purine or pyrimidine base from the sugar-phosphate backbone sensitizes DNA to alkali-induced strand scission w10x. In the absence of a base, the potential aldehyde group of the deoxyribose moiety facilitates the cleavage of the sugar 3X-phosphate bond after the alkali-catalyzed beta-elimination reaction. In contrast to alkaline pH base-free sites are relatively stable under physiologic conditions, having a half-live of at least 100 h w10,12x. However, they can be removed in cells more rapidly due to the action of AP-endonucleases w10,13x. Repair of DNA damage is an important factor for risk assessment, because, in general, longer persisting DNA alterations can be expected to have more important biological consequences than those which can be repaired effectively. No efficient repair of SL induced by cyclophosphamide was observed in SD1 cells after the medium with cyclophosphamide had been replaced by fresh medium Žwithout cyclophosphamide.. On the other hand, SL induced by ionizing radiation were repaired efficiently. The lack in repair of cyclophosphamide induced SL could be due to an inhibition of DNA repair enzymes by cyclophosphamide. However, SD1 cells treated with cyclophosphamide plus ionizing radiation repaired the amount of SL induced by ionizing radiation whereas a fraction of SL equal to those induced by cyclophosphamide alone persisted. Those experiments showed that incubation of SD1 cells with cyclophosphamide for 48 h did not inhibit the capacity of SD1 cells to repair radiation induced SL. Thus, the most probable explanation for the deficiency in repair of cyclophosphamide induced SL is that cyclophosphamide and ionizing radiation induce different types of SL. The radiation-type SL Žpredominantly direct DNA single-strand breaks. can be repaired by SD1 cells, contrary to the cyclophosphamide-type of SL Žpre-
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dominantly alkali labile sites.. However, an effective repair of cyclophosphamide-induced SL was observed in other cell types such as human leukemia cells w6x or mouse embryo cephalic tissue in vivo w19x. Crook observed a maximum level of SL in human leukemia cells 4 h after a 1 h exposure to hepatocyte-activated cyclophosphamide, which declined to control levels after 8 h w6x. Pillans observed a maximum increase in SL in embryonal cephalic tissue 9 h after exposure of pregnant C3H mice to cyclophosphamide w19x. No evidence of SL was present 22 h after exposure. Thus, SD1 cells differ from human leukemia cells and mouse embryo cephalic cells in their lack in efficient repair of cyclophosphamide induced SL. Induction of SL or DNA crosslinks by metabolites of cyclophosphamide has already been shown in earlier studies using self activating derivates of cyclophosphamide w5,15x, using hepatocytes as an activating system w6x, in animals w14,19x and in humans in vivo w20,21x. In general, the results from those studies agree to the present observations, using a recombinant cell line. Crook et al. w6x, Erickson et al. w5x, and Hilton et al. w15x observed a maximum in the level of DNA crosslinks 3–6 h after exposure of cells in vitro to cyclophosphamide-metabolites followed by a gradual decline. Some authors suggested repair of DNA crosslinks as a possible explanation w5,6x. However, the present study has shown that this explanation should be treated with caution, until it has been proven that no SL arise during the period of putative crosslink repair, because induction of SL and DNA crosslink repair influence elution rates in a similar way.The above references are not cited in the text. Please add.
Acknowledgements We thank Prof. Dr. J. Doehmer Žpresent address: Inst. Toxicol., TU Munchen, Germany. for SD1 cells ¨ ŽDoehmer, Dogra, Friedberg, Monier, Adesnik, Glatt and Oesch, Proc. Natl. Acad. Sci. USA 85: 5769– 5773, 1988. and Ms. S. Gebhard ŽInst. Toxicol., University of Mainz. for analysis of DNA strand breaks ŽHengstler, Fuchs, Gebhard and Oesch, Mutation Res. 304: 229–234, 1994.. This study was
supported by the Stiftung Rheinland-Pfalz fur ¨ Innovation.
References w1x Cox, P.J., Farmer, P.B. and Jarman, M. ŽEds.. Ž1976. Symposium on the metabolism and mechanism of action of cyclophosphamide. Cancer Treat. Rep., 60, 299–525. w2x Hill, D.L. Ž1975. A Review of Cyclophosphamide. Springfield, III.: Charles C. Thomas, Publisher. w3x Brock, N. and Hohorst, H.J. Ž1963. Ueber die Aktivierung von Cyclophosphamid in vivo und in vitro. Arzneim.-Forsch., 13, 1021–1031. w4x Doehmer, J., Seidel, A., Oesch, F. and Glatt, H.R. Ž1988. Proc. Natl. Acad. Sci. USA, 85, 5769–5773. w5x Erickson, L.C., Ramonas, L.M., Zaharko, D.S. and Kohn, K.W. Ž1980. Cytotoxicity and DNA cross-linking activity of 4-sulfidocyclophosphamides in mouse leukemia cells in vitro. Cancer Res., 40, 4216–4220. w6x Crook, T.R., Souhami, R.L. and McLean, A.E.M. Ž1986. Cytotoxicity, DNA cross-linking, and single strand breaks induced by activated cyclophosphamide and acrolein in human leukemia cells. Cancer Res., 46, 5029–5034. w7x Kohn, K.W., Ewig, R.A.G., Erickson, L.C. and Zwelling, L.A. Ž1980. Measurement of strand breaks and cross-links by alkaline elution. in: E.C. Friedberg and P.C. Hanawalt ŽEds.., DNA Repair, pp. 379–401. w8x Williams, J.R., Little, J.B. and Shipley, W.U. Ž1974. Association of mammalian cell death with a specific endonucleolytic degradation of DNA. Nature, 252, 754–755. w9x Redwood, W.R., Hilton, J., Colvin, M. and Owens, A.H. Ž1982. The cellular uptake of 4-hydroperoxycyclophosphamide. Proc. Am. Assoc. Cancer Res., 23, 169. w10x Kohn, K.W. Ž1992. Principles and practice of DNA filter elution. Pharmacol. Ther., 49, 55–77. w11x Hemminki, K. Ž1985. Binding of metabolites of cyclophosphamide to DNA in a rat liver microsomal system and in vivo in mice. Cancer Res., 45, 4237–4243. w12x Lindahl, T. and Anderson, A. Ž1972. Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry, 11, 3618–3623. w13x Moran, M.F. and Ebisuzaki, K. Ž1986. DNA repair pathway in alkylated human cells: apurinicrapyrimidinic intermediate resolved by escherichia coli endonuclease IV-coupled alkaline elution. Carcinogenesis, 5, 1231–1234. w14x Little, S.A. and Mirkes, P.E. Ž1987. DNA cross-linking and single-strand breaks induced by teratogenic concentrations of 4-hydroperoxycyclophosphamide and phosphoramide mustard in postimplantation rat embryos. Cancer Res., 47, 5421– 5426. w15x Hilton, J. Ž1984. Deoxyribonucleic acid crosslinking by 4hydroperoxycyclophosphamide in cyclophosphamidesensitive and -resistant L1210 cells. Biochem. Pharmacol., 33, 1867–1872. w19x Pillans, P.I., Ponzi, S.F. and Parker, M.I. Ž1989. Cyclophos-
J.G. Hengstler et al.r Mutation Research 373 (1997) 215–223 phamide induced DNA strand breaks in mouse embryo cephalic tissue in vivo. Carcinogenesis, 10, 83–85. w20x Hengstler, J.G., Fuchs, J. and Oesch, F. Ž1992. DNA strand breaks and DNA cross-links in peripheral mononuclear blood cells of ovarian cancer patients during chemotherapy with cyclophosphamidercarboplatin. Cancer Res., 52, 5622–5626.
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w21x DeNeve, W., Valeriote, F., Edelstein, M., Everett, C. and Bischoff, M. Ž1989. In vivo DNA cross-linking by cyclophosphamide: Comparison of human chronic lymphatic leukemia cells with mouse L1210 leukemia and normal bone marrow cells. Cancer Res., 49, 3452–3456.
Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1 J.G. Hengstler a
a,)
, A. Hengst a , J. Fuchs a , B. Tanner b, J. Pohl c , F. Oesch
a
Institute of Toxicology, UniÕersity of Mainz, Obere Zahlbacher Str. 67, 55131 Mainz, Germany b Department of Gynecology, UniÕersity of Mainz, 55131 Mainz, Germany c ASTA Medica AG, 60001 Frankfurt, Germany Received 13 June 1996; revised 15 August 1996; accepted 16 August 1996
Abstract Cyclophosphamide requires metabolic activation by cytochrome P450 to exert its genotoxic effects. Therefore in vitro studies on its mechanism of action have been limited to the use of self-activating derivatives of cyclophosphamide or to hepatocytes as an activating system. In this study we used a cell line of Chinese hamster lung fibroblasts ŽV79 cells., genetically engineered to express active cytochrome P450 2B1 as the sole observable cytochrome P450 ŽSD1 cells.. An increase in DNA strand lesions ŽSL: DNA single-strand breaks and alkali labile sites. was observed between 0.5 and 1.5 mM cyclophosphamide Ž24 h incubation. which could be classified as alkali labile sites using a modified alkaline elution assay. Compared to cyclophosphamide, its active metabolite 4-hydroperoxycyclophosphamide Ž4-OOH-CY. was about 250-fold more effective in induction of SL. Equimolar concentrations of phosphoramide mustard Ž50 mM., the ultimate DNA binding metabolite of cyclophosphamide, caused only about 50% of SL compared to 4-OOH-CY. A minimum of 12 h of incubation of SD1 cells was needed for cyclophosphamide Ž1 mM. until SL were detectable, compared to only 2 h for 4-OOH-CY and 1.5 h for phosphoramide mustard Ž50 mM.. DNA crosslinks were observable after shorter incubation periods than single-strand breaks Ž6 h for cyclophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard. and were no longer detectable at incubation periods of more than 20 h. Treatment of SD1 cells with ionizing radiation only, cyclophosphamide only, and radiation plus cyclophosphamide showed that SL induced by cyclophosphamide were not repaired during incubation with fresh culture medium Ž24 h.. However, an efficient repair of SL caused by ionizing radiation was observed and was not inhibited by cyclophosphamide. These observations give strong evidence that different types of SL were induced by cyclophosphamide and radiation. SD1 cells were able to repair the special kind of SL induced by radiation but not the SL caused by cyclophosphamide. Keywords: 4-Hydroxycyclophosphamide; Phosphoramide mustard; Cytochrome P450 2B1; DNA-repair; Alkali labile site; Ionizing radiation
1. Introduction Abbreviations: SL, DNA strand lesion ŽDNA single-strand breaks and alkali labile sites.; 4-OOH-CY, 4-hydroperoxycyclophosphamide ) Corresponding author. Tel.: q49 6131r173115; Fax: q49 6131r230506.
Cyclophosphamide is one of the most often applied antitumor agents w1,2x in clinical practice. However, studies on the mechanism of action of cyclophosphamide at a cellular level have been diffi-
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cult, because cyclophosphamide requires activation by hepatic enzymes ŽFig. 1.. Cyclophosphamide is activated by mixed-function oxidases w3,4x to 4-hydroxycyclophosphamide which spontaneously decomposes to acrolein and phosphoramide mustard. Thus, the requirement for cytochrome P450-mediated activation of cyclophosphamide has limited in vitro studies to the use of self-activating derivatives of cyclophosphamide, such as 4-hydroperoxycyclophosphamide or sulfidocyclophosphamide derivatives w5x or the use of hepatocytes as activating systems w6x. In this study we used SD1 cells, which are Chinese hamster lung fibroblasts ŽV79 cells. genetically engineered to express active cytochrome P450 2B1
as the sole detectable cytochrome P450 w4x. SD1 cells are able to activate cyclophosphamide and due to this they were used in this study to examine the induction and repair of DNA single-strand breaks as well as crosslinks induced by cyclophosphamide and its metabolites phosphoramide mustard and 4-hydroperoxycyclophosphamide Žwhich releases 4-hydroxycyclophosphamide in aqueous solution.. Additionally DNA damaging effects of cyclophosphamide in SD1 cells could be compared to those in mock transfected V79 cells, the parental cell line which does not express any detectable activity of cytochrome P450. Thus, it was possible to differentiate between those effects of cyclophosphamide which can be exerted only after activation by cytochrome P450 2B1 and those which can be induced by cyclophosphamide itself without any activation by cytochrome P450.
2. Materials and methods 2.1. Alkaline elution
Fig. 1. Metabolism of cyclophosphamide.
The alkaline elution method of Kohn et al. w7x was employed with some modifications. A suspension of 2 million cells in 1 ml of cold phosphate-buffered saline ŽPBS. was poured onto polycarbonate filters ŽNucleopore, Tubingen, Germany; 25 mm diameter, ¨ 2 mm pore size.. Cells were lysed with 3 ml of a solution of 10 mM ethylenediaminetetraacetic acid ŽEDTA., 0.5% Triton X-100, 20 M NaCl Žadjusted to pH 10 with NaOH. for 60 min. After the lysis cells were washed with 4.5 ml 10 mM EDTA Žadjusted to pH 10 with NaOH. using a flow rate of 3 mlrh for 90 min. Before starting sampling of the eluted DNA, one fraction equal to the volume of the tube Ž0.25 ml. was discarded. The elution was performed at 48C in the dark using a 5 M NaCl, 20 mM EDTA solution adjusted to pH 12.6 with NaOH. Using a pump speed of 1.5 mlrh, the eluting solution was collected over a period of 10 hrs in a single fraction. After removal of the filters, they were sonicated in 15 ml elution buffer for 2 = 15 min Žfilter fraction.. Quantification of the eluted DNA and of the DNA in the filter fractions was performed using an automated detection system Žcontinuous
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flow detection system; Skalar Analytic, Erkelenz, Germany. which was based on the following treatment: the sample was neutralized with 0.1 M KH 2 PO4 adjusted with K 2 HPO4 Ž0.1 M. to pH 6.0. Thereafter the neutralized solution was mixed with a fluorochrome solution Ž0.1 M K 2 HPO4 adjusted with KH 2 PO4 Ž0.1 M. to pH 7.2; 0.88 mgrl bisbenzimide; Riedel-de-Haen, Seelze, Germany.. The fluorescent complex was measured at an emission wavelength of 450 nm and an excitation wavelength of 365 nm. The elution rate was calculated as yŽlog 10 R . r 10 h, where R represents the fraction of DNA remaining on the filter after 10 h of elution. The described alkaline elution technique was standardized with gamma-irradiation Žcaesium source; 500 cGyrmin., which induced a linear, dose-dependent increase in DNA single-strand breaks. An increase in elution rate of 12 = 10y3 hy1 was induced by 1 Gy. Each sample was eluted on four separate filters and mean values were calculated. For the analysis of total DNA crosslinks, cells were irradiated with 800 cGy prior to elution using a cesium gamma-irradiation source Ždose rate: 500 cGyrmin.. Cells were kept on ice 15 min before and during irradiation. Sensitivity of DNA crosslink analysis was not improved if higher or lower radiation doses were used. For the analysis of the kinetics of formation of alkali labile sites, the solution eluting through the filter was collected in 12 fractions of 60 min each. Elution rates obtained by an elution solution adjusted to pH 12.6 were compared to elution rates obtained by an elution solution adjusted to pH 12.2, as the rate of conversion of alkali labile sites to DNA single-strand breaks is known to be increased with increasing pH w10x. Thus, lower DNA elution rates are observed at pH 12.2 compared to pH 12.6 if alkali labile sites are present, whereas no difference will be observed if only direct DNA single-strand breaks are present.
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SD1 cells were treated with geneticin ŽG 418; 400 mgrml. once a month to select cells transfected with cytochrome P450 2B1 w4x. However, cytotoxicity of cyclophosphamide to SD1 cells was never increased after selection with geneticin during the period of this study, which shows a relatively stable expression of cytochrome P450 2B1. Cells were split in a 1 : 2 ratio 24 h before they were incubated with drugs. Cyclophosphamide, 4-OOH-CY, and phosphoramide mustard were dissolved in Dulbecco’s minimal essential medium and added to half- confluent SD1 cells in 60-mm Petri dishes. Before analysis by alkaline elution the cells were washed with 10 ml of phosphate-buffered saline, harvested from the plate after trypsinization, and the viability was measured by trypan blue exclusion. Phosphoramide mustard and 4-OOH-CY were stored at y208C and added to the culture medium of the cells within 3 min when they had been dissolved.
4. Results A dose-dependent induction of DNA strand lesions ŽSL. by cyclophosphamide and its metabolites 4-hydroxycyclophosphamide and phosphoramide mustard was observed in SD1 cells after 24 h of incubation ŽFigs. 2 and 3.. For cyclophosphamide a significant increase in SL was observed at concentrations between 0.5 and 1.5 mM ŽFig. 2.. No further increase in SL could be observed between 1.5 and 4 mM. In the dose range from 0.5–1.5 mM no statistically significant decrease in trypan blue exclusion was observed compared to control cells ŽFig. 2.. At
3. Cell culture and drug treatment SD1 cells w4x were cultured at 378C, PŽCO. 2 s 5%, with Dulbecco’s minimal essential medium supplemented with 10% foetal calf serum 125 mgrliter of streptomycin, and penicillin Ž0.125 mega unitsrliter..
Fig. 2. Induction of DNA strand lesions Želution rate. and trypan blue exclusion of SD1 cells after incubation with cyclophosphamide for 24 h. Mean values and standard deviations of three independent experiments are given.
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Fig. 3. Induction of DNA strand lesions Želution rate. in SD1 cells after incubation with 4-hydroxycyclophosphamide and phosphoramide mustard for 24 h. Mean values and standard deviations of three independent experiments are given.
concentrations higher than 4 mM a further increase in SL appeared and in the same dose range the SD1 cells lost their ability to exclude trypan blue. Compared to cyclophospham ide, 4-hydroper oxycyclophosphamide Žwhich spontaneously releases 4-hydroxycyclophosphamide . induced similar Fig. 5. Induction of DNA crosslinks Žwhich correlate with the decrease in elution rate. in SD1 cells after incubation with 1 mM hydro peroxcyclophosphamide Ža. and with 50 mM 4-hydroxycyclophosphamide or 50 mM phosphoramide mustard Žb. for various incubation periods. Mean values and standard deviations of three independent experiments are given.
Fig. 4. Induction of DNA strand lesions Želution rate. in SD1 cells after incubation with 2 mM cyclophosphamide Ža. and with 50 mM 4-hydroperoxycyclophosphamide or 50 mM phosphoramide mustard Žb. for various incubation periods. Mean values and standard deviations of three independent experiments are given.
amounts of SL if about 250-fold lower concentrations were used ŽFig. 3.. Equimolar concentrations of phosphoramide mustard caused only about 50% of SL compared to 4-OOH-CY. Up to the highest concentrations of 4-OOH-CY and phosphoramide mustard no statistically significant decrease in trypan blue exclusion was observed. A time-dependent induction of SL was observed after incubation of SD1 cells with 2 mM cyclophosphamide or 50 mM 4-OOH-CY or 50 mM phosphoramide mustard ŽFig. 4.. For cyclophosphamide a statistically significant increase in SL was observed after 12 h of incubation, but not for shorter incubation periods ŽFig. 4a.. However, 4-OOH-CY and phosphoramide mustard induced detectable amounts of SL already after 2 h of incubation ŽFig. 4b.. In order to investigate DNA crosslinks, cells were irradiated with 800 cGy after incubation with cyclophosphamide. DNA crosslinks were detectable after shorter incubation periods than SL ŽFig. 5.. For
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Fig. 6. Alkaline elution profiles of SD1 cells after incubation with 2 mM cyclophosphamide for 24 h and of control cells Ža., as well as SD1 cells after irradiation with 800 cGy and control cells Žb. using elution solutions adjusted to pH 12.2 and pH 12.6. The ordinate gives the logarithm of the percentage of DNA retained on the filters. The abscissa gives the time DNA was eluted through the filter.
cyclophosphamide Ž1 mM. an induction of DNA crosslinks Žwhich was shown by a decrease in elution rates. was observed after 6 h of incubation and reached a maximum after about 15 h. However, no induction of DNA crosslinks was observed if shorter incubation periods were used ŽFig. 5a.. Phosphoramide mustard Ž50 mM. and 4-OOH-CY Ž50 mM. induced detectable amounts of DNA crosslinks already after 1 h of incubation ŽFig. 5b.. A higher level of DNA crosslinks was induced by 4-OOH-CY compared to phosphoramide mustard. To differentiate between direct DNA single-strand breaks and alkali labile sites ŽDNA-lesions which are converted to DNA single-strand breaks by alkaline pH; the rate of conversion increases with increasing
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pH w10x. SD1 cells were incubated with cyclophosphamide Ž2 mM; 48 h. and alkaline elution profiles were compared using an elution solution adjusted to pH 12.6 and 12.2 ŽFig. 6.. SD1 cells irradiated with 800 cGy were used as a control, because ionizing radiation is known to induce predominantly direct DNA single-strand breaks. For the irradiated SD1 cells, no statistically significant difference could be observed using elution solutions adjusted to pH 12.6 and 12.2 ŽFig. 6b.. However, for SD1 cells incubated with cyclophosphamide an about 7-fold higher elution rate was detected at pH 12.6 compared to pH 12.2 ŽFig. 6a.. As the elution of the DNA from control cells was almost identical at pH 12.6 and 12.2, only the data obtained at pH 12.6 are shown in Fig. 6a and b. To examine whether SL induced by cyclophosphamide can be repaired in SD1 cells, cyclophosphamide treated SD1 cells Ž2 mM; 24 h. were washed twice with PBS, the medium containing cyclophosphamide was replaced by fresh medium Žwithout cyclophosphamide. and the cells were further incubated at 378C. No repair of SL was observed up to 3 h of postincubation ŽFig. 7.. The lack in repair of cyclophosphamide-induced SL could be due to an inhibition of DNA repair enzymes by cyclophosphamide. Alternatively SD1 cells could be constitutionally deficient in repair of SL. Therefore we examined DNA repair in SD1 cells which were only treated
Fig. 7. DNA strand lesions Želution rate. in SD1 cells after incubation with 2 mM cyclophosphamide for 24 h and subsequent incubation in fresh medium for 0.5, 1.5, and 3 h. Mean values and standard deviations of three independent experiments are given.
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riod in fresh medium Žwithout cyclophosphamide. the level of SL decreased by the amount of SL which had been induced by ratiation, but did not decline significantly below the level of SL which were induced by treatment with cyclophosphamide only.
5. Discussion
Fig. 8. DNA strand lesions in SD1 cells directly after irradiation with 600 cGy and after subsequent incubation at 378C for 30 min and 3 h Ža. and in SD1 cells after incubation with cyclophosphamide Ž2 mM; 48 h., after incubation with cyclophosphamide Ž2 mM; 48 h. and additional irradiation with 600 cGy, and after a subsequent incubation period in fresh medium without cyclophosphamide Ž378C. for 30 min and 3 h Žb.. Mean values and standard deviations of three independent experiments are given.
with gamma-radiation ŽFig. 8a. and with gammaradiation plus cyclophosphamide ŽFig. 8b.. SD1 cells only treated with 600 cGy gamma-radiation repaired about 60% of the initially induced SL after 30 min postincubation at 378C. After 3 h of incubation the level of SL had declined to the level of the controls ŽFig. 8.a.. Thus, SD1 cells could efficiently repair SL induced by ionizing radiation contrary to those induced by cyclophosphamide. Treatment of SD1 cells with 2 mM cyclophosphamide for 48 h induced an increase of SL to 340% of the control cells ŽFig. 8b.. If SD1 cells were incubated for 48 h with 2 mM cyclophosphamide and additionally irradiated with 600 cGy the elution rate increased to 780% of the control cells. During the subsequent incubation pe-
SD1 cells, which express cytochrome P450 2B1, were able to activate cyclophosphamide w4x. Thus, these cells can be used to investigate the mechanisms of cyclophosphamide toxicity and mutagenicity in the same cells in which the active metabolites are formed and do not require hepatocytes as an external activating system. Cyclophosphamide induced a dose-dependent increase in DNA strand lesions ŽSL. in SD1 cells in a dose range from 0.5–1.5 mM cyclophosphamide. From 1.5-4 mM the dose-effect curve showed no further increase, which may be due to a saturation of cytochrome P450 2B1. Up to 4 mM cyclophosphamide SD1 cells excluded trypan blue. On account of that fact, SL observed up to this concentration resulted from a direct attack to the DNA and not from indirect effects due to a loss of cell membrane integrity w8x. At concentrations higher than 4 mM SD1 cells lost their ability to exclude trypan blue. Thus, SL induced in this concentration range seem to be caused by an indirect effect due to the loss of cell membrane integrity and the release of DNA degrading enzymes from lysosomes w8x. Induction of SL by cyclophosphamide was compared to that of 4-hydroperoxycyclophosphamide Ž4OOH-CY. and phosphoramide mustard. 4-OOH-CY is transported into the cells and spontaneously releases 4-hydroxycyclophosphamide w9,14,15x. In vivo 4-hydroxycyclophosphamide is formed in the liver and transported to the tumor via the blood stream w6x. Phosphoramide mustard is considered to be the ultimate DNA binding metabolite w6x. In the present study 4-OOH-CY was about 2-fold more active in inducing SL in SD1 cells than phosphoramide mustard. The smaller effect of phosphoramide mustard is probably due to the higher polarity of phosphoramide mustard at physiological pH, which renders penetration through the cell membrane more difficult
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w5x or due to the absence of the effect of acrolein, also inducing SL w6x. 4-OOH-CY was about 250-fold more active than cyclophosphamide. Thus, only a relatively small fraction of cyclophosphamide seems to be activated to 4-OOH-CY by SD1 cells. A minimum of 12 h of incubation of SD1 cells was needed for cyclophosphamide Ž2 mM. until SL were detectable, compared to only 2 h for 4-OOH-CY Ž50 mM. and 1.5 h for phosphoramide mustard Ž50 mM.. These differences show that a relatively long period of time was needed by SD1 cells to produce an amount of genotoxic cyclophosphamide metabolites, which were sufficient to induce a detectable level of SL. On the other hand formation of phosphoramide mustard from 4-OOH-CY must be a relatively fast process. DNA strand lesions cause an increased elution of DNA through the filters during alkaline elution, whereas DNA crosslinks decrease DNA elution. On principle, DNA crosslinks should be measurable by the detection of the decreased elution of DNA from control cells through filters. However, at least 85% of the DNA from control cells is retained on the filter using the standard alkaline elution procedure. Thus, the small range from 85 to 100% retention of DNA does not allow a sensitive detection of DNA crosslinks. Owing to that fact, artificial SL are induced by ionizing radiation prior to elution to increase the sensitivity of DNA crosslink detection w10x. We found 600-800 cGy to be an optimal dose range. Using substantially higher doses the sensitivity of crosslink detection decreased again, because DNA fragments became so small that nearly all DNA eluted through the filter, irrespective of whether DNA crosslinks were present or not. In this study DNA crosslinks were detectable after shorter incubation time than SL Ž6 h for cyclophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard.. They were no longer detectable at incubation times longer than 20 h, in contrast to SL which increased up to the longest incubation times tested Ž52 h.. The observed increase in elution rates after a temporary decrease ŽFig. 5. must not be interpreted as repair of DNA crosslinks, because it occurred in the same period, in which an increase in SL was observed ŽFig. 4.. An explanation might be a conversion of crosslinks to alkali labile sites caused by a loss of purine bases as a consequence of destabilization of
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N-glycosidic bonds by bivalent adducts. This is supported by high-performance liquid chromatography analysis of DNA adducts caused by cyclophosphamide, which have shown that cyclophosphamide was able to induce crosslinks between N7 positions of guanine w11x. Those adducts depurinated readily and after 24 h at pH 7.0 and 378C 70% of them had been liberated. This agrees with the present examination, which has classified cyclophosphamide induced SL as alkali labile. Alkali lability has been shown to be typical for depurination sites w10x. The loss of a purine or pyrimidine base from the sugar-phosphate backbone sensitizes DNA to alkali-induced strand scission w10x. In the absence of a base, the potential aldehyde group of the deoxyribose moiety facilitates the cleavage of the sugar 3X-phosphate bond after the alkali-catalyzed beta-elimination reaction. In contrast to alkaline pH base-free sites are relatively stable under physiologic conditions, having a half-live of at least 100 h w10,12x. However, they can be removed in cells more rapidly due to the action of AP-endonucleases w10,13x. Repair of DNA damage is an important factor for risk assessment, because, in general, longer persisting DNA alterations can be expected to have more important biological consequences than those which can be repaired effectively. No efficient repair of SL induced by cyclophosphamide was observed in SD1 cells after the medium with cyclophosphamide had been replaced by fresh medium Žwithout cyclophosphamide.. On the other hand, SL induced by ionizing radiation were repaired efficiently. The lack in repair of cyclophosphamide induced SL could be due to an inhibition of DNA repair enzymes by cyclophosphamide. However, SD1 cells treated with cyclophosphamide plus ionizing radiation repaired the amount of SL induced by ionizing radiation whereas a fraction of SL equal to those induced by cyclophosphamide alone persisted. Those experiments showed that incubation of SD1 cells with cyclophosphamide for 48 h did not inhibit the capacity of SD1 cells to repair radiation induced SL. Thus, the most probable explanation for the deficiency in repair of cyclophosphamide induced SL is that cyclophosphamide and ionizing radiation induce different types of SL. The radiation-type SL Žpredominantly direct DNA single-strand breaks. can be repaired by SD1 cells, contrary to the cyclophosphamide-type of SL Žpre-
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dominantly alkali labile sites.. However, an effective repair of cyclophosphamide-induced SL was observed in other cell types such as human leukemia cells w6x or mouse embryo cephalic tissue in vivo w19x. Crook observed a maximum level of SL in human leukemia cells 4 h after a 1 h exposure to hepatocyte-activated cyclophosphamide, which declined to control levels after 8 h w6x. Pillans observed a maximum increase in SL in embryonal cephalic tissue 9 h after exposure of pregnant C3H mice to cyclophosphamide w19x. No evidence of SL was present 22 h after exposure. Thus, SD1 cells differ from human leukemia cells and mouse embryo cephalic cells in their lack in efficient repair of cyclophosphamide induced SL. Induction of SL or DNA crosslinks by metabolites of cyclophosphamide has already been shown in earlier studies using self activating derivates of cyclophosphamide w5,15x, using hepatocytes as an activating system w6x, in animals w14,19x and in humans in vivo w20,21x. In general, the results from those studies agree to the present observations, using a recombinant cell line. Crook et al. w6x, Erickson et al. w5x, and Hilton et al. w15x observed a maximum in the level of DNA crosslinks 3–6 h after exposure of cells in vitro to cyclophosphamide-metabolites followed by a gradual decline. Some authors suggested repair of DNA crosslinks as a possible explanation w5,6x. However, the present study has shown that this explanation should be treated with caution, until it has been proven that no SL arise during the period of putative crosslink repair, because induction of SL and DNA crosslink repair influence elution rates in a similar way.The above references are not cited in the text. Please add.
Acknowledgements We thank Prof. Dr. J. Doehmer Žpresent address: Inst. Toxicol., TU Munchen, Germany. for SD1 cells ¨ ŽDoehmer, Dogra, Friedberg, Monier, Adesnik, Glatt and Oesch, Proc. Natl. Acad. Sci. USA 85: 5769– 5773, 1988. and Ms. S. Gebhard ŽInst. Toxicol., University of Mainz. for analysis of DNA strand breaks ŽHengstler, Fuchs, Gebhard and Oesch, Mutation Res. 304: 229–234, 1994.. This study was
supported by the Stiftung Rheinland-Pfalz fur ¨ Innovation.
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J.G. Hengstler et al.r Mutation Research 373 (1997) 215–223 phamide induced DNA strand breaks in mouse embryo cephalic tissue in vivo. Carcinogenesis, 10, 83–85. w20x Hengstler, J.G., Fuchs, J. and Oesch, F. Ž1992. DNA strand breaks and DNA cross-links in peripheral mononuclear blood cells of ovarian cancer patients during chemotherapy with cyclophosphamidercarboplatin. Cancer Res., 52, 5622–5626.
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w21x DeNeve, W., Valeriote, F., Edelstein, M., Everett, C. and Bischoff, M. Ž1989. In vivo DNA cross-linking by cyclophosphamide: Comparison of human chronic lymphatic leukemia cells with mouse L1210 leukemia and normal bone marrow cells. Cancer Res., 49, 3452–3456.