Br. J. Cancer (1978) 37, Suppl. III, 129

SOME FACTORS AFFECTING THE SPECIFIC TOXICITY OF MISONIDAZOLE TOWARDS HYPOXIC MAMMALIAN CELLS I. J. STRATFORD* AND P. GRAY From the CRC Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN

Summary.-The toxic action of misonidazole towards hypoxic mammalian cells has been shown to be a function of serum concentration, with higher serum concentrations enhancing the toxic effect. Added thiols protect cells against misonidazole toxicity. In addition, the action of misonidazole on hypoxic cells labelled with 5-BUdR has been examined. Cells with incorporated 5-BUdR are no more sensitive to misonidazole toxicity than are cells without label.

MISONIDAZOLE is more toxic towards hypoxic than to aerobic mammalian cells in vitro (Hall and Roizin-Towle, 1975; Moore, Palcic and Skarsgard, 1976; Stratford and Adams, 1977) and in multicell spheroids (Sridhar, Koch and Sutherland, 1976). The molecular mechanism of this differential toxic effect remains unclear, although it is believed to be associated with anaerobic reduction of the nitro group in misonidazole. The experiments described in the present work were aimed at shedding some light on the likely target for misonidazole damage and the mechanism by which this damage occurs. MATERIALS AND METHODS

These were carried out in a manner similar to that described in detail elsewhere (Stratford and Adams, 1977) and only a brief outline will be given here. Spinner flasks were fitted with a gas inlet/outlet system and a sidearm through which samples could be withdrawn. Asynchronous, log phase, Chinese hamster V79-379A cells at a concentration of 5 x 105 cells/ml were suspended in MEM + 7-5% fes and held in a water bath at 37°C. The cell suspension was deaerated by flowing N2 + 5% CO2 over the surface of the stirred suspension throughout the experiment. Samples of cells for plating were withdrawn at appropriate times. For some experiments cells were labelled

with 10-5M 5-Bromodeoxyuridine (5-BUdR) for 24 h, which resulted in bifilar labelling of the DNA. After this time cells were washed free of label and prepared for toxicity experiments, as described above. From the same batch of labelled cells a sample was taken for irradiation, in order to check the enhancing action of 5-BUdR for causing radiation damage. These cells were irradiated in suspension in MEM + 7-5% fcs in air, in vessels similar to those first described by Parker, Emmerson and Skarsgard (1968). The vessels were immersed in a water bath at 370 and irradiated with 250 kVp X-rays at a dose rate of 184 rad/min. Cells with incorporated 5-BUdR were protected from light throughout the period of labelling, experimentation and subsequent colony formation (Ben-Hur and Elkind, 1972). RESULTS AND DISCUSSION

(1) Studies on cells labelled with 5-BUdR Radiation survival curves for cells with incorporated 5-BUdR are shown in Fig. 1. The survival curve for cells incubated for 24 h prior to irradiation in 10-5M thymidine is identical to untreated cells (Do= 305 rad), whereas the 5-BUdR labelled cells show a considerably enhanced sensitivity (Do 139 rad). Under these conditions of test the action of 5-BUdR appears to be entirely dose-modifying, with an extrapolation number, n 23.

* Present address: Physics Department, Institute of Cancer Research, Clifton Avenue, Sutton, Surrey SM2 5PX, U.K. 10

130

I. J. STRATFORD AND P. GRAY

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FIG. 2.-The effect of incorporated 5-BUdR on the toxicity of 2 mm misonidazole towards hypoxic mammalian cells. 0, Cells grown in spinner culture medium (SCM)

DOSE I rad

supplemented with 10-5 M 5-BUdR; 0, Cells grown in SCM plus 10-5 M thymidine; x, Cells grown in SCM.

FIG. 1.-The radiation response of Chinese hamster V79-379A cells grown in 5-BUdR for 24 h then irradiated at 37°C in air at 184 rad/min. - - - Cells grown in spinner culture medium (SCM); 0 Cells grown in Cells grown in SCM + 10-5 M 5-BIJdR; SCM + 10-5 M Thymidine.

These results contrast with the findings when cells labelled with 10-5 M 5-BUdR are exposed to 2 mm misonidazole under hypoxic conditions (Fig. 2). Under these conditions the toxic action of misonidazole towards cells with incorporated 5-BUdR is no different from that towards unlabelled cells. It has been reported that the action of drugs whose target is known to be DNA, e.g. the alkylating agents nitrogen mustard and dimethylmyleran, have their toxicity considerably enhanced by the incorporation of 5-BUdR into cells (Schindler, Ramseier and Grieder, 1966). Also Hall et al. (1977) found that Misonidazole did not produce

any

7 8 5 6 2 3 4 after noculation with 2mM Ro-07-0582 I h

.

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aberrations in hypoxic cells after a drug treatment sufficient to reduce survival to 10-2. The result in the present work may provide incidental evidence to suggest that DNA is not the primary target for

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FIG. 3.-The effect of added

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No serum;

* 5% serum; A, 20% serum; *, 50%

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CYTOTOXICITY OF MIZONIDAZOLE

131

appears to be general for compounds with free -SH. Typical data are given in Fig. 4, where hypoxic cells held in 5 mm misonidazole are treated with various concentrations of D-penicillamine. It has been suggested that misonidazole toxicity is, in part, a consequence of depletion of intracellular -SH (Hall et al., 1977), our results would add support to this suggestion. In addition, preliminary data would indicate that free -SH will protect both hypoxic and oxic cells against misonidazole toxicity (Gray and Stratford, unpublished), which may indicate that the mechanism of misonidazole toxicity is the same under aerobic and hypoxic conditions

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Tlne cells are in 5mM miswoidazole under hypoxic conditions /h FIG. 4.-The effect of D-Penicillamine on the toxicity of 5 mm misonidazole towards hypoxic mammalian cells. 0, No thiol; *, 2 mm-SH; A, 5 mM-SH.

misonidazole conditions.

toxicity

under

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(2) The effect of serum concentration and added sulphydryls on misonidazole anaerobic toxicity Added serum does not alter the radiosensitizing effectiveness of misonidazole nor does it significantly alter the absolute radiation response of V79 cells (Asquith et al., 1974). This contrasts with the findings in Fig. 3 where added serum considerably enhances the toxicity of misonidazole. We would suggest that this is not due to any protein binding but rather that the added serum provides more reducing equivalents for reduction of the nitro group in misonidazole. Hall et al. (1977) have shown that the sulphydryl, cysteamine, protects hypoxic cells against misonidazole toxicity. We have confirmed this in our system and also shown that this protective action

REFERENCES ASQUITH, J. C., WATTS, M. E., PATEL, K., SMITHEN, C. E. & ADAMS, G. E. (1974). Electron Affinic Sensitization of Hypoxic Bacteria and Mammalian Cells In vitro by some Nitroimidazoles and Nitropyrazoles. Radiat. Res., 60, 108. BEN-HUR, E. & ELKIND, M. M. (1972) Survival Response of Asynchronous and Synchronous Chinese Hamster Cells Exposed to Fluorescent Light following 5-Bromodeoxyuridine Incorporation. Mut. Re8., 14, 237. HALL, E. J., ASTOR, M., GEARD, C. & BIAGLOW, J. (1977) 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. HALL, E. J. & RoIzIN-TowLE, L. (1975) Hypoxic Sensitizers: Radiobiological Studies at the Cellular Level. Radiology, 117, 453. MOORE, B. A., PALCIC, B. & SKARSGARD, L. D. (1976) Radiosensitizing and Toxic Effects of the 2-Nitroimidazole Ro-07-0582 in Hypoxic Mammalian Cells. Radiat. Re8., 67, 459. PARKER, L., EMMERSON, P. T. & SKARSGARD, L. D. (1969) Sensitization of Anoxic Mammalian Cells by Triacetoneamine N-Oxyl. Survival and Toxicity Studies. Radiat. Res., 38, 493. SCHINDLER, R., RAMSEIER, L. & GRIEDER, A. (1966) Increased Sensitivity of Mammalian Cell Cultures to Radiomimetic Alkylating Agents following Incorporation of 5-Bromodeoxyuridine into Cellular DNA. Biochem. Pharmac., 15, 2013. SRIDHAR, R., KoCH, C. & SUTHERLAND, R. (1976) Cytotoxicity of Two Nitroimidazole Radiosensitizers in an In vitro Tumor Model. Int. J. Radiat. Onc. Biol. Phys., 1, 1149. STRATFORD, I. J. & ADAMS, G. E. (1977) Effect of Hyperthermia on Differential Cytotoxicity of a Hypoxic Cell Radiosensitizer, Ro-07-0582, on Mammalian Cells In vitro. Br. J. Cancer, 35, 307.

Some factors affecting the specific toxicity of misonidazole towards hypoxic mammalian cells.

Br. J. Cancer (1978) 37, Suppl. III, 129 SOME FACTORS AFFECTING THE SPECIFIC TOXICITY OF MISONIDAZOLE TOWARDS HYPOXIC MAMMALIAN CELLS I. J. STRATFORD...
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