a comparison of the cytological effects of three hypoxic cell radiosensitizers

In, J Kodrolwn Oncolo~v Bml Ph,w.. Vol 8. pp I207 121 5 0360.3016,‘82/071207-09503 00/O

Pnntcd ,n the U S A All r,ght\ reserved Copyright 0 1982 Pergamon Press Lid

??Resident Essay Award-1981 ASTR

A COMPARISON OF THE CYTOLOGICAL EFFECTS OF THREE HYPOXIC CELL RADIOSENSITIZERS

JEROME J. SPUNBERG, M.D.,* CHARLES R. GEARD, PH.D? AND MELANIE H.

RUTLEDGE-FREEMAN, M.S.$

Radiological Research Laboratory Department of Radiology Cancer Center/Institute of Cancer Research Columbia University College of Physicians & Surgeons New York, NY 10032

Misonidazole has entered Phase III clinical trials as a hypoxic cell radiosensitizer. Neurotoxocity is the major dose-limiting factor and has prompted the development of two further compounds with reduced lipophilicity and shorter half-life in viva. Aside from the short-term problem of neurotoxocity, other potential long-term consequences should be considered. Such is the purpose of this investigation where the cytological effects of three radiosensitizers upon oxic and hypoxic Chinese hamster V-79 cells have been examined. Two newer compounds, desmethylmisonidazole and Stanford Research compound 2508, were compared with their clinically used predecessor, misonidazole. Under aerated conditions, cell killing was increased with SR-2508 in a concentration and time dependent manner, so as to exceed by more than three times the level produced by the other two drugs at 5 mM for 72 hours. Cell progression into mitosis was also markedly reduced by as much as l/IO,000 of control values. However, as the three compounds induced similar frequencies of sister chromatid exchange (SCE) and chromosome aberration, the enhanced cytotoxic effect of SR-2508 appears to be mediated via an interphase rather than a post-mitotic cell death. Cells were made hypoxic and treated with the three drugs for 4 hr, then mitoses sequentially collected for 16 hr. The three compounds produced similar levels of cell killing, slowing of cell cycle progression, SCE’s and chromosome aberrations, with cycle-specific effect on S and G-l phase cells for SCE induction. These results indicate that desmethylmisonidazole and misonidazole have similar cytotoxic and clastogenic properties under oxic and hypoxic conditions. SR-2508 is relatively more toxic to aerated cells and may deserve close clinical observation for toxicity to normal tissues; further, all three agents may enhance DNA damage and mutagenesis in tissues that are normally hypoxic.

Sensitizers, Cytological effects, Oxic/hypoxic cells.

INTRODUCTION

Several members of the nitroimidazole class of drugs are currently undergoing clinical evaluation as hypoxic cell radiosensitizers. Misonidazole, a 2-nitroimidazole, has already reached Phase III clinical trials.24 However, the dose that may be administered is limited to below that required for maximal effect by the appearance of neurotoxicity,‘.” which has spurred further attempts to synthe- size and test new compounds with similar sensitizing properties but less restrictive side effects. Reduction of drug lipophilicity and consequent decrease in penetration into the central and peripheral nervous system is one approach to the problem,4 together with reduced half life.

Both desmethylmisonidazole and SR-2508 possess this property, which is perceived to be advantageous while maintaining indistinguishable radiosensitizing ability.”

In addition to the relatively short-term toxicity, it is essential that other more subtle factors of long-term consequence be considered, such as damage to the DNA of normal cells and carcinogenic risk. The cytotoxicity and clastogenicity of misonidazole have been well docu- mented in vitro in several mammalian cell lines, both under aerated7,X.” and hypoxich,“’ “.‘c conditions (and suggested to be comparatively safe to normal tissues at clinically relevant concentrations). One may not assume that structurally similar agents will have comparable

*Chief Resident in Radiation Therapy. ?Assistant Professor of Radiology. *Research Assistant. This investigation was supported by Contract DE-

AC0278EV04733 from the Department of Energy and by Grant No. CA I2536 to the Radiological Research Laboratory and by Grant No. CA 13696 to the Cancer Center/Institute of Cancer Research, awarded by the National Cancer Institute, DHHS.

Reprint requests to: Jerome J. Spunberg. M.D., Department of Radiation Therapy, Albert Einstein Medical Center, York and Tabor Roads, Philadelphia. PA I9 I4 I,

Acknowled~~:mmr~~Drs. Chu H. Chang and Eric .I. Ha]] provided guidance and encouragement during the course of this investigation.

Accepted for publication 25 February I 9x2.

1208 Radiation Oncology 0 Biology 0 Physics July 1982, Volume 8, Number 7

risks. SR-2508 has already been shown to be substantially less cytotoxic toward hypoxic Chinese hamster V-79 cells than misonidazole or desmethylmisonidazole.” Neverthe- less Miller and Hall” demonstrated a five to six-fold increase in transformation frequency with SR-2508 as compared with the other two agents under aerated conditions. The direct impact of these radiosensitizing drugs on DNA as determined by sister chromatid exchange (SCE) frequency, chromosomal aberrations, and changes in the progression through the cell cycle as well as direct cell killing may not only aid in the evaluation of potential clinical toxicity but also provide indications as to differing mechanisms of action based upon molecular structural changes.

minutes in 0.3 M Na,HPO, adjusted to pH 10.4 containing 2% Giemsa stain.’ SCE’s were scored in 1000 second- division chromosomes for each treatment and the frequency of first, second, and third division mitoses were assessed in 200 cell samples. Mitotic indices were determined from a minimum of 1000 cell samples, with larger samples necessary for treatments producing very low indices. As Giemsa staining is not considered adequate for chromosomal aberration analysis, these were scored from slides stained with 2% acetoorcein, with 50 cells scored per treatment.

Part 2-Hypoxic and oxic V-79 cells in suspension

METHODS AND MATERIALS

Part l-Attached aerated V-79 cells A. Cell Culture and Drug Treatment-Chinese ham-

ster V-79 cells were grown at 37.5% in 100 mm petri dishes* for chromosome studies and T-25 flasks for survival studies in culture medium?, supplemented with 10% calf serum, antibiotics, and L-glutamine. Misonida- zole$, desmethylmisonidazole$and SR-25085 were added to aerated log phase cells to concentrations of 1, 2, and 5 mM (time zero). Bromodeoxyuridine (BrdU) at 5 PM was added at time zero and 24 and 48 hours later for 24, 48, and 72 hour drug treatments respectively. Cultures were maintained in total darkness or subdued light once the BrdU had been added.

B. Cell Survival-Attached cells were exposed to 1, 2, and 5 mM of each drug for 24,48, and 72 hours, with or without 5 PM BrdU for the 24 hours preceding the conclusion of the drug treatment. At the termination of treatment, the medium was changed and survival determined by fixing, staining, and colony counting after 7-8 days incubation in total darkness. Minimum colony size was considered to be approximately 60 cells. Each treatment was compared to appropriate controls to determine the effects of drug contact alone or in the presence of BrdU.

A. Cell Culture and Drug Treatment-Cells were grown at 37.5”C in plastic flasks* as in Part 1. BrdU at 5 PM was added and allowed to become incorporated into DNA for 20 hours (about 2 replication cycles) before hypoxic treatment. Log phase cells were harvested by trypsinization, centrifuged, washed, and counted. Cell suspensions were prepared at 2 x lo6 cells/ml for hypoxic cells and 1 x 10’ cells/ml for aerated cells with or without misonidazole, desmethylmisonidazole, or SR-2508 at 1,2, or 5 mM and dispensed into labeled 1 ml glass ampules. Hypoxia was induced by the metabolic depletion tech- nique13 using high-purity nitrogen plus 5% CO* flush before heat sealing and agitation in a 37% water bath for 4 hours. Oxic cells were similarly flushed with air plus 5% CO, before heat sealing and maintenance at room tem- perature for 4 hours. At the conclusion of the 4 hour treatment, the ampules were agitated on a vortex mixer, opened, and aliquots of cells assayed for survival or chromosomal damage according to the plan of the particular experiment.

B. Cell Survival-Cells in ampules were treated with 0, 1, 2 or 5 mM misonidazole, desmethylmisonidazole, or SR-2508 under oxic or hypoxic conditions in the presence or absence of 5pM BrdU. Ampules were then emptied and aliquots diluted for plating into flasks containing fresh growth medium. After incubation for 7-8 days in total darkness, colonies were prepared and counted as in Part 1.

C. Cell Cycle and Chromosomal Effects-For each drug treatment, colcemid at 3 PM was added four hours before the completion of the assigned contact time of 24, 48, or 72 hours. Cells were harvested to determine mitotic index, SCE frequency, and chromosomal aberrations. Because of the incorporated BrdU, cultures were handled in total darkness or subdued light throughout the cell preparation period until fixation. Following trypsinization and centrifugation, cells were made hypotonic with 0.075 M KCl, fixed in cold 75-25% methanol-acetic acid, and then dropped on wet slides and air dried. Staining was performed by immersing slides for 10 to 20

C. Cell-Cycle and Chromosomal Effects-Cells with incorporated BrdU were treated in ampules as described above. Upon completion, ampules were emptied into 100 mm petri dishes containing medium and 5 PM BrdU. One ampule was used per dish for the hypoxic treatments, while 5 ampules were used per dish for the oxic treatments to ensure adequate numbers of mitoses. Mitotic cells were then sequentially accumulated with 3 /IM colcemid for 2 hr up to 16 hr after treatment. This allows an evaluation of cellular progression through the cell cycle and of cell cycle stage specific effects on SCE’s and chromosomal aberrations. Cells were prepared, fixed, stained and scored as in Part 1-C above.

*Corning. tGibco S-10.

SDonated by Hoffman-LaRoche, Nutley, N.J. $Donated by Dr. J. Martin Brown, Stanford University.

Cytological effects of three sensitizers 0 J. J. SPLNBERG et al. I709

-BrdU

\ Ro-07-0592 Ro-05-9963

z

\

5mM

\

sR2508

+ BrdU

\ ! 5mM \

SR 2508

h 48 712

Drug Contact Time (hrs)

k bs 712

Drug Contact Time (hrs) Fig. 1. Survival data for aerated Chinese hamster V-79 cells exposed for periods of time to graded doses of misonidazole (circles) desmethylmisonidazole (squares) and SR-2508 (triangles) in the presence and absence of 5 PM bromodeoxyuridinc. Standard errors are shown.

RESULTS

Part I-Attached aerated V-79 cells A. Cell survival. Figure 1 shows cell survival curves for

24, 48, and 72 hours exposure times to each of the three drugs tested, misonidazole (Ro-07-0582), desmethylmisonidazole (Ro-05-9963), and SR-2508, at 1, 2, and 5 mM concentrations. At the lower drug concentrations there is little effect of prolonged contact time on survival for all three drugs as compared with control. However, at 5 mM, SR-2508 demonstrates increased cytotoxicity with marked enhancement as contact time increases so as to exceed 3 times the effect seen with 5 mM misonidazole and desmethylmisonidazole at 72 hours.

There is no consistent enhanced cell killing in the presence of 5 PM BrdU for the final 24 hours of drug treatment at any concentration for any drug.

B. Cell cycle eflects. The proportions of mitoses relative to control for colcemid accumulations over the final 4 hours of drug treatment are shown in Figure 2 for all three drugs. Misonidazole and desmethylmisonidazole demonstrate a small similar reduction in mitoses with increasing concentrations from 1 to 5 mM, but little impact of increased contact times of 48 and 72 hours as compared with 24 hours. In sharp contrast, SR-2508 produces dramatic decreases in mitoses with increasing concentrations and drug contact times, so as to reach approximately 1 / 1000 and 1 / 10,000 the values of control

for 48 and 72 hours contact time respectively. Therefore the results show a consistent effect of SR-2508 in reduc- ing the fraction of cells moving through the cell cycle in a drug concentration and contact time dependent manner as opposed to the other two agents where a much smaller response is present.

C. Chromosomal efects. Figure 3 depicts the frequency of SCE’s per second division chromosome as a function of drug concentration and contact time for all three drugs. In each case there is a drug effect in the induction of SCE’s related to contact time with the greatest change occurring between 0 and ImM for each contact time. Higher concentrations produce minimal increases as compared to the 1 mM results. The three drugs are similar in that SCE frequency is approximately double that of control for maximal concentration and contact time in each case. The 5 mM concentration of SR-2508, however, produced an insufficient number of second division mitoses to be scored at 24 hours and none at 48 and 72 hours so that only I and 2 mM data are available for comparison. It should be noted that while 5 mM SR-2508 for 24 hr did allow some cells to progress into mitosis, none of these were second division cells. Hence as well as preventing cellular progression, SR- 2508 also increases the length of the cell cycle in cycling cells.

The percentage of cells with chromosomal aberrations


b-07-0582 0.1 I A I I I I I

I I I I

1 2 3 4 5

Drug Concentration (mM)

Fig. 2. Relative proportion of cells in mitosis after exposure of aerated Chinese hamster V-79 cells to graded doses of drugs for 24 hr (circles); 48 hr (squares) and 72 hr (triangles). Mitoses were colcemid accumulated over the last 4 hrs of treatment.

after 24, 48, and 72 hours drug treatment is summarized in Table 1 and compared with controls. Fifty cells were scored per point. There is a tendency toward increasing aberration frequency with longer drug contact times but no such relationship with drug dose beyond 1 mM concentration. Overall SR-2508 produces a greater percentage of aberrations than misonidazole or desmethylmisonidazole with, however, no cells scored at 5 mM for 48 and 72 hour contact times.

Part 2-Hypoxic and oxic V-79 cells in suspension A. Cell survival. The drug treatment time of 4 hours

under hypoxia for suspended V-79 cells was selected to permit evaluation of cell cycle progression and chromosomal effects. Longer exposures would increase cell kill-

0.71 R o-07-0582

021 , I 1

0.7 O l 2 3 4 5

SR 2508

no 2 nd

division cells after 5mM

24 hrs drug contact

0.2 1 , , I , 1 0 1 2 3 4 5

Drug Concentration (mm) Fig. 3. The frequencies of sister chromatid exchanges (SCE’s) per V-79 chromosome after aerated cell exposure to graded doses of drugs for 24 hr (circles); 48 hr (squares); or 72 hr (triangles), No 2nd division cells were allowed into mitosis after treatment with 5 mM SR-2508, while the cells from 5 mM desmethylmisonidazole for 48 hr were mishandled (asterisk). Standard errors of the mean are shown while 5 PM bromodeoxyuridine was made available to cells 20 hr prior to fixation.

ing during interphase and thereby preclude the study of SCE’s and chromosome aberrations.6.8 As expected for such short hypoxic drug treatment times,5,‘0 there was no significant difference in the cytotoxicity of the three radiation sensitizers, with approximately a 0.45 survival fraction for each at 5 mM concentration. I and 2 mM levels produced intermediate survival results. However, 4 hour oxic treatments at 5 mM drug concentrations produced survivals no different from controls. There was no

Cytological effects of three sensitizers 0 J. J. SPUNBERC et al. 121 I

Table 1. Percentage of cells with chromosomal aberrations after treatment of attached, aerated V-79 cells*

Drug contact times

24 hr 48 hr 72 hr

Control 6 4 0

mM I 14

Misonidazole 2 6 5 6

Desmethyl- I 6 misonidazole 2 10

5 4 1 8

SR 2508 2 I8 5 16

6 14 2 16 4 8 8 16

12 14 10 I8 I6 18 14 12 t t

*SO cells scored per point. ~No cells could be scored.

demonstrable BrdU effect at 5 PM for oxic or hypoxic treatments.

B. Cell cycle eflects. Figure 4 depicts the cumulative percentage of mitotic cells versus time post treatment for each drug and concentration. A straight line on such a plot indicates a constant rate of flow of cells into mitosis. After an initial delay, this can be seen to be substantially so for the control cells and while there is some additional drug induced delay at the lower concentrations, the subsequent rate of flow into mitosis is similar to the control. However the 5 mM drug concentrations as well as increasing the delay, also appear to slow the rate of progression of cells into mitosis, with desmethylmisonidazole showing the most pronounced effect

“OT

Misonidazok (Ro-074582)

Control

Hours Post-treatment

00

; I- 0.6 z

K- 0 n 0 02 0.5 i $z

cn I

Ro-05-9963

Fig. 5. Relative proportion of cells progressing into mitosis up to 16 hours after treatment of hypoxic V-79 cells with graded doses of misonidazole (circles); desmethylmisonidazole (squares) and SR-2508 (triangles).

Figure 5 depicts the relative proportion of cells progressing into mitosis over the 16 hour period following hypoxic drug treatment at 1,2, and 5 mM. All three drugs demonstrate concentration dependent suppression of mitotic index up to 5 mM concentration compared to control. However desmethylmisondazole is more effective in that only 50% of the number of untreated cells enter mitosis at 5 mM as opposed to 70% for the other two drugs.

Hours Post-treatment Hours Post-treatment

Fig. 4. Cumulative frequencies of mitotic cells after treatment of hypoxic V-79 cells with I mM (square): 2 mM (triangles) and 5 mM (inverted triangles) misonidazole, desmethylmisonidazole and SR-2508. Mitoses were sequentially colcemid accumulated over 2 hour intervals for 16 hr post treatment. A straight line on such a plot indicates a constant rate of Row of cells into mitosis.


The relative frequencies of first, second, and third C. Chromosomal effects. The frequency of SCE’s per division mitoses over the 16 hour post-treatment period second division chromosome as a function of time follow- (using 2 hour colcemid accumulations) are shown in ing drug treatment is shown in Figure 7 for oxic and Figure 6 for the three drugs at 5 mM concentrations and hypoxic cells exposed to 0 and 5 mM drug concentration. control. The 1 and 2 mM concentrations had lesser There is no difference between hypoxic treated cells and effects. All three agents produce a reduced rate of flow control over the first 10 hours. Later, however, there is a through the cycle with less than 20% of mitoses being marked similar enhancement of SCE frequency for all third division at 16 hours compared to the 40% value for three drugs at 5mM rising to a level of 40% above control control. As before, desmethylmisonidazole yields the by 16 hours. The curve suggests a cell cycle-specific effect greatest reduction in the rate of cell cycle progression in the S and G- 1 phases, the points in the cell cycle where with no third division mitoses appearing at all until the cells would have been lo-16 hours earlier at the time of 12-14 hour collection period versus the S-10 hour period drug treatment. Values for I and 2 mM concentrations for the control. fell intermediate between control and 5 mM levels.

In all cases 1st division cells appeared in all collection periods. This indicates that a fraction of the cell popula- tion is cycling much slower than the majority of cells. This was also true for the cells treated under oxic conditions (data not shown) and may reflect an effect of the ampule technique, which is however enhanced by drug treatment.

These results imply both a drug-induced reduction in the number of cycling cells as seen in Figures 4 and 5, and a delay in the rate of cell cycle progression for those cells that are actively cycling in Figure 6. While all three drugs display these effects, desmethylmisonidazole appears to exceed misonidazole and SR-2508 in the magnitude of the effect.

Figure 8 compares SCE frequency during the 0 to 10 hour post-treatment period with the 10 to 16 hour period as a function of drug concentration. No concentration effect is seen over the first 10 hours, but over the following 6 hours a direct relationship is present between SCE frequency per second division chromosome and concentration of drug. Thus, over the 6 hour period when an impact of drug treatment appears in Figure 7, there is also a concentration dependency from 1 to 5 mM. The concept of cycle-specific effect of the drugs on S and G-l phase cells is thereby supported. As with the cell cycle studies

5 MM SR 2505 5 mM drug concentrations

Hours Post Treatment Fig. 6. Histograms depicting the frequencies of mitoses in their first division after bromodeoxyuridine incorporation (left axis) and at their third division after bromodeoxyuridine incorporation (right axis and dark hatching) with time post hypoxic cell treatment. The majority of cells were second divisions (light hatching), while histograms are shown separately for control and 5 mM misonidazole, desmethylmisonidazole and SR-2508.

Hypoxic cells

0a3 i 1 Oxic cells

0.2.1, 0 2 4 6 8 10 12 14 16

Hours Post Treatment Fig. 7. Frequencies of sister chromatid exchanges per V-79 chromosome at 2 hourly fixation intervals after hypoxic (upper panel) and oxic (lower panel) cell treatments. Controls (open circles); 5 mM misonidazole (solid circles); 5 mM desmethylmisonidazole (squares) and 5 mM SR-2508 (triangles) are shown separately with their standard errors.

Cytological effects of three semitilers 0 J. J. SPU~BER~; rr al. 1213

??? ?u

4 Ir / lo-16 hrs

/

5 m%!_ 0 / ti q o-*-a- i * 0 -10 hrs

0.4_ Post hypoxic treatment , 1 1 I

0 1 2 3 4 5 6

Drug Concentration (mM) Fig. 8. Mean frequencies of sister chromatid exchanges per V-79 chromosome versus drug concentration for the periods O-IO and IO-1 6 hr after treatment of hypoxic cells. Control (open circles); misonidazole (solid circles); desmethylmisonidazole (squares) and SR-2508 (triangles) are separately shown, while the standard errors are approximately the same size as the symbols.

there is a tendency for desmethylmisonidazole to show greater effects both under oxic and hypoxic conditions.

Table 2 lists the percentage of cells with chromosomal aberrations for the I6 hour period following the 4 hour hypoxic 5 mM drug treatments. Results are based upon 50 cells scored per point. No dose, time or drug relationship is present, although all three drugs produce approximately a doubling in the frequency of aberrations overall. This lack of cell-cycle specific effect is distinct from the results of the SCE evaluation above.

DISCUSSION

Normal tissue toxicity limits the utility of drugs administered as hypoxic cell radiosensitizers. Midonidazole has already been extensively tested for cytotoxic and clasto- genie properties in multiple in vitro cell lines and deemed to be comparatively safe to normal tissue at the clinically

Table 2. Percentage of cells with chromosomal aberrations after a 4 hour treatment of hypoxic V-79 cells*

Time post

treatment 5mM

Control Misonidazole

o-2 2 6 4 6 2-4 4 2 6 4 4-6 2 6 8 I2 6-8 6 6 8 8 S-IO 0 I2 6 2

IO-12 6 8 8 4 12-14 2 6 4 8 14-16 4 6 6 2

Overall 3.3 6.5 6.3 5.8

5mM Desmethyl- 5mM

misonidazole SR-2508

*50 cells scored per point.

relevant concentrations of I mM or less for contact time of 24 hours.h ‘L”.* Serious neurotoxicity in vivo, however, has prevented the administration of doses required to achieve desirable levels of radiation enhancement.5,20,‘3 Recent light and electron microscopic observations have attributed the etiology to cytoplasmic effects including damage to the cell and nuclear membranes.h,‘9 Therefore the development of newer compounds with diminished lipophilicity and shorter biologic half-life has become essential to the radiosenisitizer program for clinical use.

Desmethylmisonidazole and SR-2508 are two more recently synthesized 2-nitroimidazoles, which have been shown to be equivalent to misonidazole in sensitizing ability,” but have not yet been thoroughly tested for their potentially more subtle cytotoxic and mutagenic effects upon normal oxic and hypoxic tissues. Misonidazole and desmethylmisonidazole have been shown to have similar direct cytotoxicity in hypoxic V-79 and CHO cells at 2 and 2.5 mM concentrations respectively.‘0,‘5 SR-2508 has also been evaluated in hypoxic V-79 cells at 2mM and 5 mM concentrations and found to produce appreciably less cell killing than misonidazole after 6 hours of drug contact.*.‘” At 4 hours, however, there was little difference among the three agents. Our study confirms the similarity in cell survival at such short hypoxic treatment times.

The results in attached aerated V-79 cells are in sharp contrast to the effects under hypoxia. SR-2508 at 5 mM induces up to 3 times greater cell killing than misonidazole or desmethylmisonidazole in a time-dependent manner from 24 to 72 hours drug contact time. At the more clinically relevant lower concentrations no such differ- ences appear to exist. SR-2508 also produces a much greater reduction in mitotic index with a direct time and concentration relationship. However the frequencies of SCE’s and chromosome aberrations were similar for all three drugs with no points that could be scored for 5mM SR-2508 because of lack of second division mitoses. There is, however, no correlation between the frequencies of SCE’s and of chromosomal aberrations. The marked cell killing and low mitotic rate of SR-2508 in the absence of elevated chromosome aberrations suggests that the enhanced cytotoxic effect is mediated via an interphase rather than a post-mitotic cell death. This finding concurs with previously reported conclusions about misonidazole.’ Close observation is therefore desirable in the clinic for the potential toxicity of SR-2508 to well-oxygenated normal tissues.

The cytotoxicity of misonidazole under hypoxic conditions has also been reported to be primarily an induced interphase cell death.6 Based upon the present results, desmethylmisonidazole and SR-2508 appear to have a similar mechanism of action. The concentration-dependent reduced mitotic index coupled with the low rate of chromosomal aberrations implies that most cells are being killed before mitosis. The cells that are able to divide arrive at mitosis relatively free of chromosomal aberrations and are then unlikely to experience a post-


mitotic cell death. In addition, the reduced rate of progression through the cell cycle, as demonstrated by the diminished frequency of early third division mitoses in drug-treated cells, indicates a delay in cell cycle time for those cells surviving interphase. These toxic effects would be expected to increase with longer than 4 hour hypoxic- cell-drug interaction as might be seen in the clinical situation depending upon biologic half-life.

the body, such as the spermatogonia in the testes, may have deleterious consequences and merit clinical study.

The incidence of SCE’s is regarded as a highly sensitive indicator of mutagenic activity.*’ Known carcinogenic agents have been shown to produce an SCE frequency up to 10 times that found in controls.‘“‘26 In particular, many cancer chemotherapeutic drugs have SCE frequencies 2 to 8 times control values.” Misonidazole at clinically relevant concentrations does not produce an increase in SCE frequency greater than twice the control level, so that it is considered to be an inefficient mutagenic- carcinogenic agent.7.*,2’ Our results demonstrate that both desmethylmisonidazole and SR-2508 are comparable to misonidazole in their tendency to produce SCE’s in the presence or absence of oxygen. However under hypoxic conditions there is a clear enhancement of SCE induction for cells exposed in S and G-l phase judging from the results of sequential 2 hour colcemid accumulations. This contrasts with the cell cycle stage non-specificity for cell killing previously reported for misonidazole.6.‘4 The mechanisms behind the interphase cell killing and S and G-l phase SCE induction are therefore quite distinct. The potential mutagenic effects in normally hypoxic cells of

Miller and HallI showed that misonidazole effectively produces in vitro oncogenic transformations. Transfor- mation frequencies are similar to levels seen with drugs that are either equivocal or not associated with carcino- genesis in V~VO.~ However, SR-2508 at 1 mM was recently found to produce a transformation incidence five to six times larger than either misonidazole or desmethylmisonidazole.15

The similar levels of SCE induction in V-79 cells in the present study for all three drugs suggest that the differential in transformation frequency is a result of more than a mutagenic effect on DNA alone. Alternatively, differ- ences in response in the 2 cell lines, C3H lOT$ vs. V-79 could account for the somewhat discrepant findings.

In conclusion, desmethylmisonidazole and misonidazole have similar cytotoxic properties under oxic and hypoxic conditions. SR-2508, however, produces much more cell-killing in aerated cells and therefore deserves close clinical observation for toxicity to normal tissues. In addition, since all three agents cause more chromosomal alterations in hypoxic cells in a dose-dependent manner, especially in S and G-l phase, DNA damage and mutagenesis may be enhanced in tissues that are normally hypoxic. Clinical trials to evaluate toxicity are necessary in order to determine the potential short and long-term consequences of large-scale use of these newer hypoxic cell radiosenisitizers.

REFERENCES I Alves, P., Jonasson, J.: New staining method for the detec-

tion of sister chromatid exchanges in BrdU labelled chromosomes. J. Cell&-i. 32: 18.5-195, 1978.

2. Astor, M.B., Hall, E.J., Biaglow, J.E., Parham, J.C.: Newly synthesized hypoxia-mediated drugs as radiosensitizers and cytotoxic agents. Intl. J. Radiat. Oncol. Biol. Phys 8: 75-83, 1982.

3. Benedict, W.F., Banerjee, A., Gardner, A., Jones, P.A.: Induction of morphological transformation in mouse C3H/ lOT$ clone 8 cells and chromosomal damage in hamster a (Tl ) Cl-3 cells by cancer chemotherapeutic agents. Cancer Res. 37: 2202-2208, 1977.

4. Brown, J.M., Lee, W.W.: Pharmacokinetic considerations in radiosensitizer development. In Radiation Sensitizers, Cancer Management, Vol. 5, L. Brady (Ed.). NY, Masson. 1980, pp. 2-13.

5. Dische, S., Saunders, M.l., Lee, M.E., Adams, G.E., Flock- hart, I.R.: Clinical testing of the radiosensitizer Ro-07- 0582. Experience with multiple doses. Br. J. Cancer 34: 567-579, 1977.

6. Geard, C.R., Povlas, S.F., Astor, M.B., Hall, E.J.: Cytologi- cal effects of I -(2-nitro- 1 -imidazolyl)-3-methoxy-2-propa- nol (misonidazole) on hypoxic mammalian cells in vitro. Cancer Res. 38: 644-649, 1978.

7. Geard, C.R., Rutledge-Freeman, M.H.: Effects of long contact times of misonidazole on attached Chinese hamster V-79 cells. Mutation Res. 78: 289-294, 1980.

8. Geard, C.R., Schwartz, L.E., Rutledge-Freeman, M.H.: Clastogenic effects of misonidazole on aerated and hypoxic cells in vitro. In Radiation Sensitizers, Cancer Manage-

ment, Vol. 5, L. Brady (Ed.). NY, Masson. 1980, pp. 460-465.

9. Gray, A.J., Dische, S., Adams, G.E., Flockhart, I.R., Foster, J.L.: Clinical testing of the radiosensitizer Ro- 07-0582. 1. Dose tolerance, serum and tumour concentrations. Clin. Radiol. 27: 15 1-l 57, 1976.

IO. Hall, E.J.. Astor, M.: Comparison of sensitizers in vitro. In Radiation Sensitizers, Cancer Management, Vol. 5, L. Brady (Ed.). NY, Masson. 1980, pp. 186-190.

11. Hall, E.J., Astor, M.. Geard, C.R., Biaglow, J.: On the cytotoxicity of the hypoxic cell radiosensitizer, Ro-07-0582, the effect of hyperthermia and the reversal of the cytotoxic effect with cysteamine. Br. J. Cancer 35: 809-815, 1977.

12. Hall, E.J., Biaglow, J.: Ro-07-0582 as a radiosensitizer and cytotoxic agent. Int. J. Radiat. Oncol. Biol. Phys. 2: 521-530, 1977.

13. Hall, E.J., Roizin-Towle, L.: Hypoxic sensitizers: radiobio- logical studies at the cellular level. Radiology 117: 453- 457,1975.

14. Miller, R.C., Hall, E.J.: Oncogenic transformation in vitro by the hypoxic cell sensitizer misonidazole. Br. J. Cancer 38: 41 l-417, 1978.

15. Miller, R.C., Hall, E.J.: Oncogenic transformation in vitro produced by misonidazole. In Radiation Sensitizers, Can- cer Management, Vol. 5, L. Brady (Ed.). NY, Masson, 1980, pp. 146-151.

16. Moore, D.A., Palcic, B., Skarsgard, L.D.: Radiosensitizing and toxic effects of the 2-nitroimidazole Ro-07-0582 in hypoxic mammalian cells. Radiation Res. 67: 459-473, 1976.

Cytological effects of three sensitizers 0 J. J. SPUNBERG Ed al. 1215

17.

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a comparison of the cytological effects of three hypoxic cell radiosensitizers

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