Effects of temperature on chemically induced sister-chromatid exchange in human lymphocytes

Mutation Research, 174 (1986) 15-20

15

Elsevier

MRLett 0821

Effects of temperature on chemically induced sister-chromatid exchange in human lymphocytes Kunihiko Miura*, Kanehisa Morimoto and Akira Koizumi Department of Public Health, Faculty of Medicine, Univerity of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113 (Japan) (Accepted 25 November 1985)

Summary Lymphocytes from healthy adults were studied for sister-chromatid exchanges (SCEs) when pulse-treated in Go with mitomycin C (MMC), ethyl methanesulfonate (EMS), or 4-nitroquinoline N-oxide (4NQO) at various temperatures ranging from 0°C to 41°C and then cultured in medium containing 5-bromodeoxyuridine at 37°C. The results showed that the frequencies of SCEs induced by MMC or EMS varied according to the treatment temperature. In MMC- or EMS-exposed cultures, the SCE frequency increased continuously with increasing treatment temperature; treatment at 37°C resulted in a 3-4 times greater induction of SCEs than did that at room temperature (25°C). On the other hand, SCE frequencies in cells exposed to 4NQO remained within normal deviation, showing no temperature-dependent changes. Baseline SCE frequencies remained almost constant within the temperature range tested. These da~a indicate that treatment temperature is a very critical factor in determining the sensitivity of cells to the chemical induction of SCEs.

Lymphocytes in the circulating blood are in their resting (Go) phase of the cell cycle before stimulation by a mitogen such as phytohemagglutinin (PHA) in vitro. Recently, in many in vitro sensitivity tests using lymphocytes, cells were treated with chemicals in the Go phase to simulate conditions in vivo (Ishii and Bender, 1978; Littlefield et al., 1980, 1981; Iijima et al., 1984; Morimoto et al., 1984). It is generally recognized that the temperature during treatment is one of the most critical factors in the interaction between chemicals and biomacromolecules, including DNA, because *To whom correspondence should be addressed.

the binding between chemicals and biomacromolecules is a chemical reaction that is regulated by the reaction temperature. Furthermore because some of the chemicals require metabolic activation, the activity of the activating enzyme(s) is also influenced by the temperature. No detailed data, however, are available on the effects of temperature on the sister-chromatid exchange (SCE) frequencies in lymphocytes treated with chemicals in Go. In the present study, we investigated the effects of temperature on the SCE frequencies in Go human lymphocytes treated with chemicals at 0-41°C. We show that the SCEs induced by

0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

16 mitomycin C (MMC) or ethyl methanesulfonate (EMS) increased continuously with increasing treatment temperature, whereas SCEs induced by 4-nitroquinoline N-oxide (4NQO) showed no temperature dependency in repeated experiments. Materials and methods

treatments, the cells were centrifuged at 1500 rpm (500 × g) for 5 min and washed 3 times with PBS prewarmed to 37°C. Cells from control cultures were also washed at the same time, and the pelleted cells in 0.5 ml of PBS were then resuspended in complete culture medium (4.5 ml) containing P H A to stimulate lymphocytes to enter cell cycling.

Lymphocyte culture

Scoring

Whole blood (0.3 ml) f r o m healthy adults was added to 4.7 ml of R P M I 1640 medium containing 15070 fetal bovine serum (Gibco), 3070 P H A - M (Difco), and 40 #M bromodeoxyuridine (Sigma). The cultures were incubated at 37°C in complete darkness for 72 h. Colcemid (2 × 10 - 7 M; Calbiochem) was added to each culture 6 h before fixation.

200 Metaphase cells were scored for cellproliferative kinetics, and 30 second-division metaphase cells from each culture were scored blindly on coded slides for SCEs.

Slide preparation and sister-chromatid differential staining The cells were collected by centrifugation, exposed to 0.075 M KCI hypotonic solution for 15 min to spread the chromosomes and hemolyze the erythrocytes, and fixed in methanol:acetic acid (3:1). Drops of a concentrated suspension of ceils were placed on microslides, which were allowed to air-dry. Slides were stained by a modification o f the fluorescence-plus-Giemsa method to obtain harlequin chromosomes (Morimoto, 1983). Cells dividing for the 1st (X0, 2nd (X2), or 3rd or more (X3 +) time in culture can be determined in such preparations (Tice et al., 1976; Morimoto and Wolff, 1980).

Chemical treatment Whole blood (0.3 ml) was added to serum-free medium (4.7 ml) precooled or prewarmed in a water bath at 0, 10, 25, 33, 37 or 41°C. For treatments, M M C (Sigma) was first dissolved in distilled water, EMS (Tokyo-Kasei) or 4 N Q O (Tokyo-Kasei) in dimethyl sulfoxide. After necessary dilutions with phosphate-buffered saline (PBS), aliquots of these freshly made solutions were added to each culture to give the appropriate final concentrations. Cells were treated for 2 h at various temperatures (0-41°C). To terminate the

Results and discussion To investigate the possibility of the disturbance o f cell cycle progression, cultures f r o m one blood donor (donor B in Table 2) were incubated for 2 h at various temperatures ranging from 0°C to 41°C immediately before P H A stimulation. The results showed no consistent cell cycle disturbance as to the Go incubation temperature (Table 1). When lymphocytes in Go were exposed to M M C or EMS at various treatment temperatures, a clear curvilinear increase in SCE frequencies with increasing treatment temperature was observed (Fig. la, b and Table 2). For example, in cultures treated with M M C at 10°C, little increase in SCE frequen-

TABLE 1 BASELINE SCE FREQUENCIES AND PERCENTAGE OF FIRST (X0, SECOND (X2), AND THIRD OR SUBSEQUENT (X3 +) METAPHASE CELLS THAT HAD BEEN CULTURED FOR 2 h AT DIFFERENT TEMPERATURES IMMEDIATELY BEFORE PHA STIMULATION Temperature (°C)

SCEs/per cell

0 10 25 33 37 41

8.40 8.30 9.10 11.18 8.57 8.83

a Mean ± S.E.

+ 0.62a + 0.44 ± 0.55 + 0.76 + 0.39 _+ 0.57

% of total metaphases Xl

X2

X3+

10.7 7.5 10.2 9.1 9.2 12.7

44.3 29.5 41.4 26.7 35.9 30.3

45.0 63.0 48.4 64.2 54.9 57.0

9.27 9.10 9.60 9.64 9.74 9.34

Expl. 3 A 0 10 25 33 37 41

± ± + ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

0.53 0.60 0.66 0.72 0_6l 0.65

0.59 0.81 0.73 0.70 0.57 0.64

0.79 0.49 0.66 0.73 1.04 1.04

0.62 0.44 0.55 0.76 0.39 0.57

0.70~ 0.62 0.66 0.68 0.58 0.47

11.87 13.60 18.52 27.97 38.57 42.43

(30) (30) (30) (30) (30) (30)

(30) (30) (30) (30) (30) (30)

(30) (30) (30) (30) (30) (30)

22.60 30.47 39.47 46.07 54.47 63.33

± ± ± ± ± ±

1.60 1.99 2.02 1.75 2.17 2.48

0.68 0.74 0.85 0.98 1.48 1.43

__* 0.59 ± 0.75 _+ 0.77 ± 1.11 ± 1.30 ± 1.41

(30) (30) (30) (30) (30) (30)

(30) (30) (30) (30) (30) (30)

(30) (30) (30) (30) (30) (30)

8.40 9.43 10.83 15.17 16.50 18.90

9.17 10.73 13.40 15.05 17.20 19.77 ± ± ± ± ± ±

0.59 0.58 0.57 0.83 0.95 0.87

+ 0.61 ± 0.66 _+ 0.89 ± 1.02 ± 0.68 ± 0.89

5 × 10-3M

1.5 × 10 6 M

(30) 9.80 ± (30) 11.52 ± (30) 17.40 ± (11) 14.07 + (30) 40.30 ± (30) 43.23 ±

(30) b (30) (30) (30) (30) (30)

EMS

MMC

Mean ± S.E. of 30 cells per point. b Number of cells observed in parentheses. No second metaphase observed.

8.37 7.60 7.57 10.37 8,47 8.64

8.60 7.80 7.67 8.93 8.53 9.40

Expt. 2 C 0 l0 25 33 37 41

0 10 25 33 37 41

8.40 8.30 9.10 11.18 8.57 8.83

0 10 25 33 37 41

C

8.23 12.87 10.40 I1.10 8.10 10.17

Expt. I A 0 10 25 33 37 41

Donor Temp. Treatment (°C) Control

9.07 12.63 15.97 24.40 30.70 29.57

(30) 8.37 (30) 9.63 (30) 13.07 (30) 15.70 (30) 30.87 (30) -¢

(30) (30) (30) (30) (30) (30) ± ± ± + ±

± ± ± ± ± ± 0.62 0.50 0.67 0.77 1.18

0.68 0.88 0.68 1.16 1.14 1.23

1 × 10-ZM

(30) (30) (30) (30) (30)

(30) (30) (30) (30) (30) (7)

-

-

8.60 9.80 13.07 31.93

± ± ± ±

0.82 1.07 1.03 1.70

2 × 10-2M

+_ + ± ± ± ± ± ± ± ± _ ±

10,74 10.84 11.77 13.14 12.40 14.i7

±

± ± ± ± ±

13.27 14.20 15.10 14.57 12.80 17.50

11.33

7.27 9.93 9.07 11.20 10.93

1.63 0.70 0.91 0,89 0.68 1.14

1.09 0.78 1.06 1.11 0.83 1.23

0.88

1.21 0.50 0.84 1.14 0.83

3 x 10-6M

4NQO

(30) (30) (30) (30) (30) (30)

(30) (30) (30) (30) (30) (30)

(30)

(30) (30) (30) (30) (30)

TEMPERATURES

(30) (30) (30) (29)

SCE FREQUENCIES IN L Y M P H O C Y T E S T R E A T E D W I T H MMC, EMS OR 4NQO A T VARIOUS T R E A T M E N T

TABLE 2

22.47 26.17 22.67 19.40 19.10

± ± ± + ±

± ± ± ± ±

± + ± ± ± ±

20.13 19.93 14.63 21.67 22.80 20.83

26.40 28.74 25.37 24.17 22.17

_+ +_ ± ± ± ±

17.77 15.63 14.90 22.23 23.43 22.03

1.07 1.20 1.24 0.95 1.15

1.42 1.59 1,77 1.40 1.14

0.72 0.95 0.74 1.23 0.89 1.32

1.09 1.09 0.64 1.28 1.41 1.07

2 × 10 5

(30) (30) (30) (30) (30)

(30) (30) (30) (30) (30)

(30) (30) (30) {30) (30) (26)

(30) (30) (30) (30) (30) (30)

18

I

I

i

I

I

a.

30

/. i

i

I

I

20 (J

10 (n Z < "I" 0

I b.

60 0

50

40 tN

30

20

10

0

•

.....

:_ ......

1

I

I

I

I

0

10

20

30

40

TREATMENT TEMPERATURE (*C)

Fig. 1. SCE frequenciesin lymphocytesexposed in Go phase to 1 × 10-2M EMS(a) or 1.5 x 10-6M MMC(b) f o r 2 h a t various temperatures. Responses of donor A (O) and donor B (4,) in Expt. 1 are shown in the figure. Bars, S.E.; dashed line, mean of the individual mean baseline SCE frequenciesat each treatment temperature. cies compared with untreated cultures was observed, whereas the identical treatment at 41°C induced more than 40 SCEs (Fig. lb). In EMS-treated cells, a similar temperature dependencey in SCE frequencies was observed at the 3 EMS doses used; at higher treatment temperatures (37°C or 41°C) 2 x 10- 2 M EMS was too toxic for cells to reach second division (Table 2). In 4NQO-exposed cultures, however, cells showed no consistent increase in SCE frequency in relation to the treatment temperature in repeated experiments (Table 2), Mean baseline SCE frequencies remained within the range of 7.5-13, and no temperature dependency was observed in untreated cells from 3 donors in the repeated experiments (Table 2).

The relationship between baseline SCE frequencies and incubation temperature has already been investigated in human lymphocytes (Abdel-Fadil et al., 1982; Pandita, 1983; Das and Sharma, 1984), in cultured mammalian cells (Kato, 1980; Speit, 1980), and in plant cells (Speit, 1980; Guti6rrez et al., 1981). These studies showed that both higher and lower culture temperatures than the optimal temperature resulted in the elevation of baseline SCE frequencies. Speit (1980) also studied the influence of growth temperature on MMC-induced SCE frequencies in cultured mammalian cells; the results were quite complicated because baseline SCE frequencies showed the minimum value at 37°C, whereas MMC-induced SCEs peaked at the same temperature. Change in the treatment temperature, however, disturbs both the duration of DNA synthesis (S phase), which might result in the increase of baseline SCE frequencies (Kato, 1974), and the production of SCE-inducing cellular lesions by chemicals. Speit's results seemed to show the interaction between these two effects, whereas our study clearly excludes the possibility of a change in the duration of S phase because the change in temperature was for only 2 h in the noncycling Go phase; growth temperature was maintained at 37°C during incubation in both treated and untreated cultures. Our results, showing the effects of temperature on the formation of the SCE-inducing damage, showed a parallel increase in SCE frequencies with the increase in treatment temperature in MMC- or EMS-treated cells, and they are consistent with the results of Littlefield et al. (1981). It has been reported that some DNA-damaging agents require enzymatic metabolic activation to react with DNA. For example, it is necessary for MMC to be changed into its reduced form to have two active sites in the molecular structure, which are thought to form a crosslinking bridge within the DNA molecule (Iyer and Szybalski, 1964; Tomasz et al., 1974). The activities of cellular enzymes, however, could be influenced by a change in temperature. Our results on the MMC-induced SCE frequencies suggest that changes in the ability of enzymes to metabolically activate MMC might

19 be one of the major factors that brought about the increase in SCEs with higher treatment temperature. SCE frequencies in cells treated with 4NQO, however, varied in a rather complicated manner. We used 3 blood donors in the repeated experiments. Treatments with MMC or EMS generally showed a temperature dependency in SCE response among the donors, whereas the responses of donors A and C to 4NQO treatment showed no temperature dependency in the repeated experiments (Table 2). It is reported that 4NQO requires reduction to its active form before binding to the D N A molecule (Sugimura et al., 1966; Tada and Tada, 1972), but the present study suggests that the activation pathway o f 4NQO might be more complicated than that of MMC. Some subtle balance between activation and subsequent detoxication of 4NQO might have caused the temperatureindependent results in our experiments. This indicates the complexity of the kinetics of the interaction between cells and this agent. Recently, many studies have concentrated on the effects of hyperthermia on cell killing when combined with radiation or chemical treatment (for review, see C o n n o r et al., 1977). It is generally recognized that, in mammalian cells, hyperthermic treatment is much more effective above 43°C than that under 43°C (Bhuyan, 1979). Treatments below 43°C, however, are reported to have some effects on the cell membrane structure (Hackenbrock et al., 1976; Lepock, 1982). If the 'fluidity' of the cell membrane increases with the temperature, a resulting increase in the incorporation of DNA-damaging agents might explain the elevated SCE frequencies in cultures treated with MMC or EMS in the present study. Correspondingly, it is reported that phase transition of the lipids in cell membranes may occur below about 10°C. In this cage, the 'fluidity' of the cell membrane is expected to decrease and might cause the decrease in SCE frequencies at lower treatment temperatures, though this cannot explain the temperature independency in SCEs in cells treated with 4NQO.

In summary, we have demonstrated that SCE frequencies in lymphocytes treated in Go with MMC or EMS increase remarkably with increasing treatment temperature, whereas in 4NQO~exposed cultures SCE frequencies show no significant dependence on treatment temperature.

Acknowledgements We thank R. Yamada and Y. Katayama for excellent technical assistance. Work was supported by grants-in-aid !from the Ministry of Education, Science and CUlture of Japan, and by an award from the Nissan Science Foundation.

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