Chlorine under seige.

We have compared the actions of ADM and mAMSA in Chinese hamster V79 cells in vitro, using cell survival and sister-chromatid exchange as end-points. Equimolar concentrations of ADM and mAMSA show similar toxicities to exponentially growing cells, and both drugs are less effective in killing chronically hypoxic and plateau-phase cells. Cytotoxicity to thermotolerant cells (41°C for 16 h previously) shows little difference from that for exponential cells. Pre-treating cells with misonidazole under hypoxic conditions reduces the toxicity of both ADM and mAMSA. In addition, an ADM-resistant Chinese hamster cell line, 77A-177, was cross-resistant to mAMSA. Finally, low equimolar sub-toxic doses of both drugs were found to cause similar increases in the levels of sister-chromatid exchanges in V79 cells. These results reveal no major difference in activity between ADM and mAMSA in vitro. ADRIAMYCIN (ADM) is thought to be cytotoxic due to its ability to intercalate between adjacent base pairs in DNA (Zunino et al., 1972). It has been used clinically since 1969 and has been shown to be effective in a variety of human tumours. However, the efficacy of this drug is limited by its cardiotoxicity (Carter, 1975). Another drug which is thought to act by intercalation of DNA is 4'-1 (9

tially growing cells, and both drugs are less effective in killing chronically hypoxic and plateau-phase cells. Cytotoxicity to thermotolerant cells (41°C for 16 h previously) shows little difference from that for exponential cells. Pre-treating cells with misonidazole under hypoxic conditions reduces the toxicity of both ADM and mAMSA. In addition, an ADM-resistant Chinese hamster cell line, 77A-177, was crossresistant to mAMSA. Finally, low equimolar sub-toxic doses of both drugs were found to cause similar increases in the levels of sister-chromatid exchanges in V79 cells. These results reveal no major difference in activity between ADM and mAMSA in vitro. ADRIAMYCIN (ADM) is thought to be cytotoxic due to its ability to intercalate between adjacent base pairs in DNA (Zunino et al., 1972). It has been used clinically since 1969 and has been shown to be effective in a variety of human tumours. However, the efficacy of this drug is limited by its cardiotoxicity (Carter, 1975). Another drug which is thought to act by intercalation of DNA is 4'-1 (9acridinyl) -amino]methanesulphonmanisidide (mAMSA, NSC 249992) (Gormley et al., 1978). This acridine derivative, which was one of a series synthesized by Atwell et al. (1972), has recently completed Phase I and II clinical studies (Legha et al., 1978(Legha et al., , 1979Schneider et al., 1979Schneider et al., , 1980 and is now undergoing prospective randomized trials. Cell-kinetic studies in vitro have shown ADM and mAMSA to have similar effects, which has led to the suggestion that mAMSA may be an alternative to ADM in the clinic . The present work was undertaken to compare directly the toxicity of ADM and mAMSA to mammalian cells in vitro. This was done using V79 cells with differing culture histories and a cell line with induced resistance to ADM. The response of cells to ADM and mAMSA after treatment with misonidazole was also examined, since in previous studies where misonidazole has been used in combination with alkylating agents a synergistic cytotoxicity has been seen (Rose et al., 1980;Stratford et al., 1980). Finally, sister-chromatid exchanges (SCEs) were used to indicate DNA damage caused by each drug.

MATERIALS AND METHODS
Cells.-Chinese hamster V79-379A cells wvere used in most of these experiments. Cells were routinely grown at 37°C in 250ml conical flasks in Eagle's minimal essential medium (MEM) modified for suspension cultures (Flow Laboratories Ltd) and supplemented with 7-5o foetal calf serum (FCS) (Gibco-Biocult Ltd). Cells were maintained in logphase growth at concentrations varying between 105 and 106 cells per ml. A doubling time of 10-12 h required cells to be diluted daily. V79-379A cells were also grown in monolayer culture in MEM plus 15% FCS, and passaged twice weekly. All medium containing cells wvas buffered with bicarbonate to pH 7-4. For most experiments cells were harvested from asynchronous exponentially growing cultures.
Some experiments -were done with cells with a history of chronic hypoxia, plateauphase cells, cells cultured at pH 6-8 or thermotolerant cells. Cells w-ith a history of chronic hypoxia were prepared by taking log-phase cells and holding them for 16 h under anaerobic conditions (Smith et al., 1980;Rajaratnam et al., 1981). Plateau-phase cells were taken from unfed suspension cultures which became density inhibited at a cell concentration of 1 2-1-6 x 106 cells/ml. Under these conditions, the pH of the culture medium had been reduced to around 6-9. Other experiments involving reduced pH were done with exponentially growing cells which had been cultured in medium at pH 6-8 for 16 h before drug treatment. Thermotolerant cells were prepared by maintaining log-phase cells at 41°C (pH 7 4) for 16 h (Stratford et al., 1981b). V79 cells cultured under conditions of either reduced pH or 41TC are still capable of progression, with doubling times about 16 and 20 h respectively.
The culture conditions described above were not inimical to our V79 cells, and all these cell populations had plating efficiencies > 9000 before treatment with cytotoxic drugs.
An ADM-resistant Chinese hamster cell line (77A-177, Belli, 1979) was sometimes also studied. These cells had originally been exposed to 0-1~tM ADM for 77 weeks, followed by a further 177 weeks growth in drug-free medium, during which time they retained their resistance to ADM. In our experiments they w-ere maintained in exponential phase in monolayer culture in MEM plus 15% FCS.
Cytotoxicity studies-Cells at 5 x 105/ml were transferred to plastic universal containers with the required amount of drug to give a final volume of 10 ml. The containers were placed in a shaking water bath at 37°C in air. After 1 h the cells wrere removed, washed by centrifugation and resuspension, counted, diluted and plated in MEM plus 15% FCS. They were then incubated for 7 days at 37°C before staining in methylene blue and scoring for colony formatioin. All colonies visible at x 2 magnification ( 50 cells or more) were counted. Some experiments involved pre-treatment with misonidazole (Stratford et al., 1980). Exponentially growing cells were exposed to 5mM misonidazole at 37°C for 2 h under hypoxic conditions, before treatment in air with mAMSA or ADM.
The 77A-177 (ADM-resistant) cells were treated with ADM or mAMSA as follows. Cells w%vere harvested from a monolayer by washing with 0 02% EDTA and 0)08% trypsin, counted, diluted and plated on to 6cm plastic Petri dishes in a total volume of 2 ml MEM plus 15O% FCS. The cells were left to attach for 2-1 h, the medium aspirated from the dishes and replaced mith 2 ml fresh medium containing either mAMSA or ADM. Dishes were incubated for 1 h at 37°C, aspirated, replenished with 2 ml fresh growth medium and incubated for 7 days. As a control, parallel experiments were conducted in an identical fa.shion using V79-379A wNere harvested by trypsinization, resuspended in 0-075m KCI for 15 min and fixed with methanol:glacial acetic acid (3:1). Air-dried slides were stained in 0 4 mg/ml Hoechst 33258 for 4 h, rinsed in distilled water and mounted in 0-16M disodium orthophosphate and 0-04M trisodium citrate (pH 8-8). The slides were exposed for 45 min to fluorescent light from a Philips CS200W-4 mercury lamp mounted on a Reichert "Polyvar" microscope and filtered with a Reichert "V2" excitation filter. Finally, the slides were rinsed and restained in 40o Giemsa for 5 min.
The survival curve for mAMSA shows that for each increment in drug dose the corresponding decrease in survival is smaller. The shape of the survival curve for ADM is basically the same as for mAMSA, except that at low doses there is an apparent shoulder. Similar results for exponentially growing V79 cells exposed to ADM or mAMSA have been obtained by  and Wilson et al. (1981). Figs 2 and 3 show the cytotoxicity of ADM and mAMSA to plateau-phase cells, cells rendered chronically hypoxic, thermo-tolerant cells and cells grown at pH 6-8. When compared to data for exponentially growing cells at pH 7-4 ( Fig. 1) it is evident that, for treatment with either drug: 1. Plateau-phase cells and cells rendered chronically hypoxic before drug treatment in air are resistant to drug action; 2. After prolonged exposure of exponentially growing cells to reduced medium pH, the cytotoxicity is reduced, but this is not sufficient to account wholly for the decreased sensitivity of plateauphase cells; and 3. There is no change in the drug sensitivity of thermotolerant cells. Fig. 4 shows results comparing the effect of the two drugs on the ADM-resistant 77A-177 cells. It is clear that this cell line is cross-resistant to mAMSA.
We have carried out experiments to ascertain whether misonidazole potentiates the effect of the intercalating agents ADM and mAMSA in the same way as with alkylating agents (Rose et al., 1980;Stratford et al., 1980). whereas for 1 OtkM mAMSA survival is increased 100-fold. The cytotoxicity of ADM or mAMSA was unaffected, whether the pretreatment was with misonidazole in air for 2 h or with N2 alone for 2 h (data not shown). A comparison of the DNA-damaging effects of ADM and mAMSA was carried out using SCE as an end-point. The frequencies of SCE as a function of subtoxic doses of the drugs are summarized in the Table, and dose-response curves are illustrated in Fig. 6. Fig. 7 shows a chromosome spread for a cell treated with ]0,M BrdU for 18 h; SCE were scored by counting the exchange sites (arrows in Fig. 7). Fig 8 and 9 show the distribution of SCE per cell within the dividing population after treatment with either ADM or mAMSA. There is a dose-dependent increase in the incidence of SCE after treatment with either drug, though at higher concentrations ADM appears to be slightly more effective. SCE experiments involved cells being treated with 10uM BrdU for 18 h. In drug-free controls it was found that this treatment alone reduced the surviving fraction to -0-8. However, little further reduction in the surviving fraction is seen when a lh exposure to 041 and 0 2Mm of either drug is given in combination with BrdU treatment.

DISCUSSION
In this study we have set out to compare in vitro the cytotoxic and DNA-damaging effects of two intercalating agents, ADM and mAMSA. Both drugs produce survival curves of similar shape for exponentially growing cells. It is not clear from our data whether the shape of the survival curves is due to heterogeneity of drug sensitivities. However, it has been suggested by others (Belli & Piro, 1977) that at high concentrations ADM may saturate target binding sites. This model is consistent with the shapes of the dose-response curves for ADM, and a similar mechanism may apply to mAMSA.
There is considerable difference between the drug sensitivities of plateau-phase and log-phase cells. For 1O,UM ADM, surviving fractions are 0-4 x 10-4 and 0-25 for exponentially growing and plateau-phase cells respectively, and for a similar concentration of mAMSA the surviving fraction of these cell populations are 0-2 x 10-3 and 0-2 x 10-1. This indicates that the differential in sensitivity between cycling and non-cycling cells may be somewhat greater for ADM. It has been shown previously that there is little difference in the sensitivity of cycling and non-cycling cells towards mAMSA when iso-leucine starvation is used to produce non-cycling cultures .
Further, Tobey et al. (1976) showed that the differential toxicity of ADM for cycling vs non-cycling cells (produced by iso-leucine starvation) is much less than that reported here and in the work of others (Barranco et al., 1974;Twentyman, 1976). Non-cycling cells produced by Tobey et al. (1976Tobey et al. ( , 1978  in this work may represent different growth-arrested states. Cells harvested from exponentially growing cultures and held for 16 h under hypoxic conditions experience growth arrest, and become resistant to ADM (Smith et al., 1980). These cells are also more resistant than exponentially growing cells to mAMSA. Cells held at 41°C for long enough to modify their response to heat damage (Stratford et al., 1981a) or the cytotoxic action of some drugs (Stratford et al., 1981b) continue cycling, and this in turn is reflected in these cells having similar sensitivities to ADM and mAMSA when compared to exponentially growing cells held constantly at 37°C. Taken together these results are consistent with the proposal that the growth status of cells in vitro will be an important determinant of sensitivity to ADM or mAMSA. 55 Cells held at pH 6-8 for 16 h before drug treatment continue cycling. However, the cells at this pH are slightly more resistant to both ADM and mAMSA when compared to exponentially growing cells held at pH 7-4. Both mAMSA and ADM are weak bases, with pKa values of 7-3 (Wilson et al., 1981) and8-2 (Skovsgaard, 1977) respectively. Changes in drug uptake with associated changes in toxicity have been reported as a function of pH for cells exposed to ADM (Skovsgaard, 1977). Further, the cytotoxicity of mAMSA has been reported to be maximal at pH 7-2 (Wilson et al., 1981). The pH range 6-8-7'4 spans that in which tumour cells may be found in vivo and it may be important that at these values of pH the cytotoxic actions of ADM and mAMSA are similar.
The basic similarities between the cytotoxic effects of ADM and mAMSA are further demonstrated by the fact that a cell line with induced resistance to ADM shows cross-resistance to mAMSA. This cell line has also been shown to be crossresistant to Actinomycin D  which has been attributed to genetically controlled membrane changes resulting in decreased drug uptake .
Cells given 5mM misonidazole for 2 h in N2 before exposure to either ADM or mAMSA become resistant to the cytotoxic actions of both intercalating agents.
There is no such effect if cells are pretreated with 5mM misonidazole in air for 2 h, nor when cells are given N2 for 2 h with no misonidazole. The exposure to misonidazole in N2 reduces survival to 0-6; it is known that misonidazole shows cell-cycle specificity, in its cytotoxic action with cells in early S being most sensitive and late S/G2 cells most resistant (Whitmore & Gulyas, 1980;Stratford, 1980). The published data on the age responses of cells to ADM and mAMSA have yielded differing results (Kim & Kim, 1972;Barranco, 1975;Bhuyan et al., 1980;Deaven et al., 1978;Roberts & Millar, 1980). However, preliminary work with synchronized V79 cells (West, unpublished)  has indicated that both intercalating agents may be most effective in early S. Therefore, our results may indicate that the observed "resistance" to ADM and mAMSA is due to a cell-cycle redistribution caused by misonidazole pretreatment in N2. The somewhat greater resistance to mAMSA than to ADM may be due to subtle differences in age response between these two drugs. An alternative explanation for these results would be that the exposure of cells to misonidazole in N2 alters the cells' membrane properties or metabolic state. Such changes are known when cells are given 5mm misonidazole for 14 h in air, and these cells also become more resistant to ADM (J. Belli, personal communication). However, at present we are not able to state categorically which of these mechanisms is operating when cells are given ADM or mAMSA immediately after hypoxic exposure to 5mM misonidazole.
Previous studies have shown that ADM and other intercalating agents can profoundly increase the frequency of SCE in CHO cells (Au et al., 1981;Galloway & Wolff, 1979;Perry & Evans, 1975). Raj & Heddle (1980) have demonstrated that, in general, intercalating agents are efficient inducers of SCE, and the present work confirms these results for V79 cells exposed to ADM. Concentrations of ADM and mAMSA between 0 05 and 0-2 ttM have been used to follow the induction of SCE. Although these drug doses produce no significant cytotoxicity as measured by the clonogenic assay, we cannot, on the strength of these data alone, deduce whether this end-point may be related to SCE induction. However, it is apparent that the magnitude of SCE formation by each drug is similar (e.g. at 0u1 4uM the SCE per cell is 15-6 + 0-7 and 16-6 + 0 5 for mAMSA and ADM respectively, compared to only 6-7 + 0 3 for untreated controls). Less SCE was found in cells exposed to 015/UM or 0'2iLM mAMSA than in those treated with the equivalent concentration of ADM (Fig. 6). The data illustrated in Figs 8 and 9 show that at these drug levels there is a wide distribution of the numbers of SCE scored, with no well-defined mode, after 0-2,UM mAMSA. This change in the form of the distribution may result from slight variation in cycle-stage sensitivity to drug action, and therefore the difference between ADM and mAMSA at this dose level may not be significant. proportion of non-differentially stained mitoses (1st divisions) appeared higher after treatment with 0 2,tM mAMSA than after the corresponding dose of ADM, suggesting that at these doses mAMSA may induce more mitotic delay than ADM. Taking these qualifications into account, it is nevertheless apparent that ADM and mAMSA induce SCE at a similar frequency.
In conclusion, we have demonstrated that the DNA damaging and cytotoxic effects of ADM and mAMSA are broadly similar, under a variety of test conditions in vitro. We suggest, therefore, that should any difference in effect be seen for these drugs in vivo or indeed clinically, this difference will have its origins in the pharmacology of the two drugs, i.e. their uptake, tumour penetration, metabolism and excretion, rather than in inherent differences in cell sensitivity.
We wish to thank Dr Jim Belli for generously providing the ADM-resistant cell line; Drs W.
Wilson, G. Whitmore and R. Hill for giving us considerable unpublished information; Professor G. Adams and Dr S. Revell for many helpful discussions, and Christine Williamson for excellent technical assistance. The provision of postgraduate studentships (CW and ES) by the MRC is gratefully acknowledged.