Cytotoxicity and mutagencity of hyperthermia for diploid human lymphoblasts

J. Thermal Biology,

1977. VoL X

pp.

95 to 99. Pergamon Press. Printed in Great Britain

CYTOTOXICITY A N D M U T A G E N I C I T Y O F HYPERTHERMIA FOR D I P L O I D H U M A N LYMPHOBLASTS MtCBAEL Z. GILMAN AND WILLIAM G. TH1LLY Toxicology Group, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A.

(Received 21 January 1977)

Abstract--Time dependent survival of diploid human iymphoblasts has been measured at 42, 43. 44, 45, 46 and 47°C. Do was found to be 1500, 23, 4, 2, 1 and 0.6rain, respectively. Mutation to 6-thioguanine resistance was measured at 45~C and found to increase monotonically with time. Thus, relatively mild heat trauma can result in genetic change in somatic human cells.

INTRODUCTION

hyperthermic treatment. Heat-induced mutation has been reported in yeast and other microbial systems (Zamenhoff &Greer, 1958; Seheaberg-Frascino & Moustacchi, 1972). However, similar phenomena have not been demonstrated in mammalian systems. Our assay employs resistance to the purine analog 6-thioguanine (6TG) as a genetic marker. This change reflects mutation at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. Using this system, we have quantitatively measured the cytotoxic and mutagenic effects in diploid human lymphoblasts of mild heat treatment.

EXPOSURE to heat above normal body temperature is a common experience of humans in eating, working or in medical therapy. However, relatively little is known about the effects of heat at the cellular level in temperature ranges below those which cause visible damage. Interest was generated in the precise biological effects of heat in the range 40-50°C, when it was discovered that exposure to 42°C was selectively toxic to cancer cells (Cavaliere et ai., 1967). Subsequent studies established this selective toxicity in both human and rodent tissues (Shreck, 1966; Giovanella et al., 1973; Kim et al., 1974; Miura & Usami, 1974; Kase & Hahn, 1975 and others). MATERIALS AND METHODS Studies in the range of 40-47°C have shown that A clonal derivative of the PGLC-33 line. originally cells are resistant to short periods of exposure. The surviving fraction decreases as an exponential func- isolated by Phillip R. Glade, University of Miami, tion of exposure time for longer exposure periods. and designated by us, MIT-2, was used in all experiThe length of the period in which little cell death ments. Stock cultures were maintained in continuous is observed and the time constant of cell killing for exponential phase at 37°C (doubling time, 16.8 hours) longer exposures are both dependent on the particu- by daily dilution with antibiotic-free RPMI 1640 lar temperature used (Westra & Dewey, 1971; Palzer medium supplemented to 10~ with fetal calf serum. & Heidelberger, 1973; Harisladis et aL, 1975). Regular chromosome counts indicate that greater Several precise sites of heat action have been sug- than 80% of the cells have 46 chromosomes. Mycogested. One well studied example is the degradation plasma tests were performed at 4-month intervals by of ribosomal RNA at temperatures in the range of an independent laboratory (Microbiological Associ42-50°C (Rosenthal & Iandolo, 1970; Tomlins & ates, Bethesda, Md.) and have been consistently negaOrdal, 1971; Gray et al., 1973; Pouchelet, 1974). tive. Cell survival in the presence and absence of 6TG HeLa cell polyribosomes disaggregate during incubation at 42°C (McCormick & Penman, 1969). Waroc- was measured by cloning in soft agar (Coffmo et al., quiet & Scherrer (1969) found that no new functional 1972) over a confluent feeder layer of human fibroribosomal RNA appears in HeLa cells incubated at blasts derived from a patient with Lesch-Nyhan syn42°C and suggested the existence of a temperature- drome (Sato et al., 1972).. Mutant fraction was determined by several indesensitive step in the biosynthesis of ribosomal RNA. Direct effects of heat on DNA have also been pendent samplings, beginning 14 days post-treatmeat. observed. Exposure to 52°C leads to single-strand The 14 day post-treatment period is required to breaks in E. coli (Bridges et al., 1969a, b; Sedgwick assure full expression of the mutant phenotype (Thilly & Bridges, 1972; Woodcock & Grigg, 1972). Strand et al., 1976; Penman & Thilly, 1976). breakage does not occur in vitro, suggesting that an In reconstruction experiments previously reported enzymatic mechanism is involved (Sedgwick & (Thilly et al., 1976), it was shown that the specific Bridges, 1972). Enhanced DNA degradation by mild protocol reported here exerts no selective pressure heat treatments (41-42°C) in conjunction with radi- with regard to the presence or absence of HGPRT ation damage has been demonstrated in rodent cells activity. In addition, all clones examined, which grow (Ben-Hur et ai., 1972; Ben-Hur & Elldnd, 1974). in the presence of 10/~g/ml 6TG have 1% or less of We have investigated the possibility of direct soma- normal HGPRT activity. Mutant fraction is detertic mutation in human cells, resulting from mild mined by the ratio of the plating efficiency in the 95

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MICHAEL Z. GILMAN AND WILLIAM G. THILLY

presence of 10/~g/ml 6TG to the plating efficiency in the absence of the drug. Colonies large enough for automatic counting (Artek Systems, Farmingdale, N.Y.) appeared within 2 wks. For heat treatment experiments, the cells were centrifuged and resuspended at a density of 2 x l0 T cells/ml. One milliliter aliquots of this suspension were placed in sterile Pasteur pipettes, previously heat-sealed at one end. Due to the relatively small wall thickness of the pipettes and the large surface area to volume ratio, speedy heat transfer was attained; treated cells reached the experimental ternperature within 15 seconds. The cells were placed in constant temperature water baths (Juchheim Labortechnik, Schwartzwald, West Germany) and held to +0.1°C. Thermometers were calibrated by us from known crystal melting points. Cells were maintained at 37°C when not at hyperthermic temperatures. Control and heated cultures remained in high-density suspension for the same length of time. Sufficient numbers of cells were treated to assure the survival of at least 100 mutants in order to assure statistical reliability. After treatment, cells were suspended in fresh medium at 4 x l0 s cells/ml. Each culture was split into duplicate cultures and maintained in screwcap bottles in a humidified incubator (5% CO2, 37°Cg Cell survival was determined 24 hr after heat treatment by quadruplicate plating. Cultures were sampled and counted daily, using a Model B Coulter Counter (Coulter Electronics, Hialeah, Florida). Cultures demonstrating cell number increases were diluted to 2 x l0 s cells/ml with fresh medium. When culture densities dropped below 2 x 10~ cells/ml, cultures were centrifuged and resuspended at 4 x l0 s cells/ml. Thus, growing cultures were maintained in exponential growth and "nongrowing" cultures were maintained in a condition conducive to continuous division of the remaining viable cells. All treated cultures demonstrated exponential growth prior to the 14 day period required for phenotypic expression of 6GT resistance.

RESULTS

The survival of human lymphoblasts exposed to temperatures of 43-47°C for varying time periods is plotted in Fig. 1. Each point on the curves represents 3 or 4 replicate determinations within a single experiment. The 45°C curve represents the average of 5 independent experiments, which were highly reproducible. Exposure to 42°C for 24 hr yielded a 30% survival (data not shown). All temperature studies yielded a classic sigmoidal response, i.e., a shoulder at low doses followed by log-linear response. There was no strong evidence from the curves for the existence of small heat-resistant populations, nor for a sensitive population. In some experiments, toxicity was characterized for longer periods than shown in Fig. 1, and it continued to show log-linear kinetics, Plating efficiencies for controls in all experiments aver-

aged 3o°/0. In order to compare the effects of the different temperatures, the concept of Do was borrowed from radiobiology. Do is here defined as the time (minutes) of exposure at each temperature required to reduce

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Fig. 1. Survival of human lymphoblast line MIT-2 exposed to temperatures 43--47°C. Each experimental point represents the mean of 3-4 replicate determinations. Toxicity curve at 45° is averaged from 5 independent experiments. Cells were treated at high density and re-suspended in fresh medium immediately thereafter. Controls remained at high density at 37° for a period equivalent to the treatment period. Plating efliciencies of controls averaged about 30%, Standard errors are uniformly less than 100 of mean values. survival by l/e in the log-linear portion of the survival curve. These data are plotted in Fig. 2. Growth of treated cultures was monitored for 2-3 wks. The growth response was as expected in all cases, i.e., a period in which no increase in cell number could be detected by particle counting followed by continuous exponential growth (Fig. 3). In some treated cultures, an estimated division delay of up to five generations was observed (Table 1). The cells surviving treatment did not resume exponential division immediately after the treatment. Rather, the number of clone-forming units (surviving cells) appears to have remained constant for a period of several generations before increasing again. By assuming uniform growth of the surviving cells in a cuRure, an estimate of the surviving fraction can be made by back-extrapolating the growth curve to time zero (Fig. 3). The length of division delay can then be obtained by comparing this estimate with the known surviving fraction as calculated from the toxicity plating. If no division dday has taken place, the two methods should yield the same surviving fraction. When the actual number of surviving clone-forming units is greater than the value arrived at by extrapolation, a division delay has occurred. The length of the delay may be approximated by the time following treatment at which the extrapolation intersects the known surviving fraction, i.e., the time at which

Hyperthermia for diploid human lymphoblasts

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Fig. 2. Toxicity of exposure to temperatures 42-47°C to human lymphoblast line MIT-2 plotted as a function of temperature. Do is the time of exposure at each temperature required to reduce survival by l/e in the log-linear portion of the survival curve calculated by least squares method from data of Fig. 1. the surviving cells appear to begin dividing again (Fig.

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Fig. 3. Growth of cultures exposed to 45°C for the time periods indicated. Horizontal lines ( - - - - ) mark the calculated surviving cell population (survival × initial cell concentration). The intersection on the time axis of these lines with the appropriate back-extrapolation from exponential culture growth ( ) is used as an estimate of the growth delay induced by treatment, vertical lines ( - - - - ) (see Table 1).

3). Alternatively, the division delay in generations can be arrived at by directly comparing the two different survival estimates (Table 1). The number of doublings required to bring the apparent surviving fraction (determined by extrapolation) back up to the known value (from plating) is the length of the delay in generations. We chose 45°C as a convenient temperature at which to study heat-induced mutagenesis. The results

of three independent experiments at 45°C are plotted in Fig. 4. Each point represents the mean of a series of 1-3 mutant fraction determinations performed at 2-3 day intervals, 14-22 days after treatment. Mutant fraction was determined as the ratio of plating efficiency in the presence of 6GT to plating efficiency in the absence of 6GT. Since exposure to 45°C does not select for mutants in preference to wild-type cells (data not shown), it seems clear that significant

Table 1. Estimated growth delay induced in cultures of human lymphoblasts treated for 10 or 15 rain at 45°C (three independent experiments) Relative survival (cells/ml x 10 -3) Exp. No. l0 min exposure: 49 60 80 15 min exposure: 49 60 80

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Surviving fraction was determined by direct plating in soft agar. Relative survival is the initial surviving population in cells/ml as calculated from plating data and by back-extrapolation of the log-linear portion of the culture growth curve (Fig. 3). Growth delay in generations was estimated as log2 of the ratio of the direct plating estimate of relative survival to the extrapolation estimate.

98

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00597) and the MTT. Undergraduate Research Opportunities Program. Mrs. Michele Verba Wong provided technical assistance.

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REFERENCES o x

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Fig. 4. Toxicity (O) and mutagenicity (O, r-l, A) of exposure to 45°C to human lymphoblast line MIT-2. Toxicity is re-plotted from Fig. 1. Three independent mutagenicity experiments are plotted. Each point represents the mean of 1-3 separate mutant fraction determinations performed at 2-3 day intervals 14-22 days after treatment. Each mutant fraction determination was performed in quadruplicate. Standard errors for individual mutant fraction determinations are uniformly less than 200 of mean values.

mutant fractions were induced by 10 and 15min exposures to 45°C. DISCUSSION

The time-dependent response of human lymphoblast survival reported here is in good agreement with that reported for the human line, HeLa (Palzer & Heidelberger, 1973) and for Chinese hamster cells, (Westra & Dewey, 1971; Harisiadis et al., 1975). The dependence of toxicity (Do) on temperature (Fig. 2) is virtually identical to Suit & Schwayder's (1974) replotting of Crile's (1962) data. Our findings with regard to human cell mutation are somewhat surprising to us. Three independent experiments have shown that exposure to 45°C is decidedly mutagenic to human lymphoblasts. Since heat induced mutation has not been previously reported in human or other mammalian cells, we are unable to make comparisons with the work of other laboratories. Earlier work in our laboratory however, has shown that induction of a mutant fraction of 1 x 10 -4 by chemical mutagens, such as methynitrosourea, methynitronitroso guanidine, ICR 191 and halogenated pyrimidines, requires concentrations resulting in marked decreases in cell survival. In comparison to these "strong" mutagens, 45°C is approximately equipotent. The apparent genetic effects represent indirect evidence for heat action on DNA. The nature of this action is presently unknown. Preliminary experiments examining the mutagenicity of lower temperatures indicate that mutations are also induced at 43 and 44°C, but time dependence has not yet been defined. Acknowledoements--The research reported here was supported, in part, by grants from the National Cancer Institute (NCI 5-R01 CK 15010-02 ET), the National Institute of Environmental Health Sciences (NIEHS 5 P01 E5

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