Toxicology Letters, 53 (1990) 307-3 13

Elsevier

307

TOXLET 02447

Studies of the rad-equivalence of ethylene oxide in the presence and absence of 12-0- tetradecanoylphorbol- 13-acetate (TPA) in C3H/ lOT1/2 cells

Ada Kolman, Maria NZslund and Siv Osterman-Golkar

Department of Radiobiology, Stockholm University, Stockholm (Sweden)

(Received 10 February 1990)

(Revision received 2 1 May 1990)

(Accepted 22 May 1990)

Key words: Ethylene oxide; Rad-equivalence; 12-O-tetradecanoyl-phorbol-13-acetate (TPA)

SUMMARY

Cell transformation in vitro of C3H/lOTl/2 cells, using gamma-radiation and ethylene oxide (EtO), in

both the absence and presence of the cancer promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), was

studied. TPA promotes transformation of C3H/lOTl/2 cells to the same extent. In the dose ranges studied

the average enhancement of the transformation frequency was 2.4 and 2.5 for Et0 and gamma-radiation,

respectively. The rad-equivalence of Et0 in the presence of TPA was calculated to be 75 k 52 rad/mMh

(95% confidence interval) which is consistent with the value 78 + 14 rad/mMh (95% confidence interval)

obtained without TPA treatment.

INTRODUCTION

Neoplastic transformation in vitro, using the C3H/lOT1/2 cell system [l], has been studied in many laboratories using ultraviolet light, ionizing radiation (X- and gamma-rays, fission-spectrum neutrons) and numerous chemicals as transforming agents [2-71. A multistage process of cell transformation in vitro, comprising initia- tion and promotion has been proposed, but the mechanisms are still insufficiently understood. A role for gene mutations in proto-oncogenes as well as the possible par-

Address for correspondence: Ada Kolman, Department of Radiobiology, Stockholm University, S-106 91

Stockholm, Sweden

0378-4274/90/S 3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

308

ticipation of epigenetic mechanisms in the appearance of transformed phenotypes

was proposed [S, 91.

The influence of the cancer promoter, 12-0-tetradecanoyl-phorbol-13-acetate

(TPA), on cell transformation in C3H/lOT1/2 cells both by radiation [l&13] and by

chemicals [ 12, 14, 151 has been studied. So far, several mechanisms of tumor promo-

tion have been proposed [16]. It was recently shown [17] that TPA is responsible for

the activation of an enzyme, protein kinase C, serving as the cellular receptor for

TPA. The consequences include changes in cell membrane function, alteration of gene

expression, as well as changes in cell division, cell differentiation and proliferative

capacity.

Ethylene oxide (EtO) has been used as a model compound in our studies aiming

at the development of a methodology for risk assessment of carcinogenic chemicals

(initiators). This compound is one of the few electrophiles that occur in occupational

exposure situations without concomitant exposure to other genotoxic agents, i.e. it

is possible to check an estimated risk epidemiologically.

The dose-risk relationship for the carcinogenic action of an initiator is influenced

by promotive and co-carcinogenic conditions. The risk model proposed by Ehren-

berg [ 181 is based on the determination of the effectiveness at low doses (defined in

terms of target dose) of the test chemical and of sparsely ionizing radiation to induce

a defined genetic damage in the same experimental system. This gives a numerical

value (Q) which expresses the capacity of the genotoxic chemical to induce genetic

damage in terms of rad-equivalence.

The approach is based on the assumption that, at low doses, the impact of promo-

tive and co-carcinogenic factors on the fate of an initiated cell is independent of expo-

sure but is determined by other factors and is the same, irrespective of how the initia-

tion was brought about.

In order to test this assumption experimentally, the transforming effectiveness of

Et0 and gamma-radiation, respectively, was studied in C3H/lOT1/2 cells in the pres-

ence and absence of the cancer promoter TPA. Some data presented earlier on the

transformation frequencies in the absence of TPA [ 191 are also included in the present

report.

MATERIALS AND METHODS

Materials

Et0 was obtained from Fluka, Switzerland. 12-O-Tetradecanoylphorbol- 13-ace-

tate (TPA) was obtained from Sigma Chemical Co., U.S.A. Dulbecco’s MEM, heat-

inactivated foetal calf serum (FCS), phosphate-buffered saline (PBS) and antibiotics

were obtained from Flow Laboratories. Scotland.

Cell culture and transformation assay

C3H/lOT1/2 mouse embryo fibroblasts were obtained from A. Meyer (Shell Re-

search Centre, Sittingbourne, England). Stock cultures (between passage 9 and 12)

309

were maintained and the transfo~ation assay was performed according to Meyer et al. [20]. The ceils were cultured as described by Kolman et al. [19]. The cells were seeded at 50 x lo3 per flask and subcultured when they reached 75% confluence.

Transformation assays were performed in 25 cm2 tissue culture flasks (Sterilin Li- mited, Feltham, England). The cells were counted in a Coulter electric counter, and seeded at 1000 cells per flask (in 4 replicates) for survival estimation and at 10000 cells per flask for the transformation assay (generally in 10 replicates); 24 h after seed- ing the cells were treated with gamma-radiation or with EtO. The medium was changed twice a week during the first 4 weeks of the experiment and then once a week during the rest of the experiment (6 weeks altogether). A lower FCS concentration (5%) was applied after the cells had reached confluence. At the end of the experiment the cells were fixed with methanol, stained with Giemsa, and foci of type II and III were analysed under the light microscope [I].

Treatment with Et0 Stock solutions of Et0 (200 mM) were prepared by weighing in PBS, in tightly

closed screw-cap tubes. The cells were treated in tightly closed tissue culture flasks for 1 h, at 37°C. The medium was removed, and the cells were rinsed once with 5 ml PBS. Fresh medium, with or without TPA, supplemented with 10% FCS, was added.

The dose of Et0 (mMh) is given as initial concentration x time of treatment [22].

The cells were irradiated with 100-400 rad (13’Cs source, model IC 900 irradiation chamber, dose-rate 72 rad/min). After treatment the medium was aspirated, and fresh medium, with or without TPA, was added,

Treatment with TPA A stock solution of TPA (1 mg/ml) in liquid-chromatography-grade acetone was

prepared and stored at -20°C. The concentration of TPA in the medium was 0.2 pg/ml in all experiments. Medium containing TPA was added immediately, at 48 h or at day 5 after exposure to Et0 or gamma-radiation, and then changed as described above.

RESULTS

TPA, added to the medium immediately, at 48 h or at day 5 after the treatment with gamma-rays or Et0 and then present in the medium during the whole experi- ment, has little or no influence on survival (data not shown).

The transformation frequencies induced by gamma-radiation and Et0 in the ab- sence and presence of TPA, added immediately after the treatment, are shown in Table I (where also the 95% confidence intervals are given).

310

TABLE I

EFFECT OF Et0 AND GAMMA-RADIATION ON THE TRANSFORMATION OF C3H/lOT1/2

CELLS IN THE ABSENCE AND PRESENCE OF TPA, ADDED IMMEDIATELY AFTER TREAT-

MENT

Treatment Total no. No. foci/

of survivors No. flasks

Mean value of transformation

frequency (foci per IO3 survivors,

with 95% confidence interval;

based on Poisson distribution)

Control 191340 141137

Control + TPA 34770 4123

EtO, 2.5 mMh 61720 31146

EtO, 2.5 mMh + TPA 40500 72135

EtO, 5.0 mMh 37460 57149

EtO, 5.0 mMh + TPA 25800 SO/30

100 rad 23230 5117

100 rad + TPA 22150 14/19

200 rad 61400 37150

200 rad + TPA 21500 40/20

400 rad 25210 40129

400 rad + TPA 12530 40119

0.07 (0.040.12)

0.12 (0.034.30)

0.50 (0.31W.71)

1.78 (1.39-2.24)

1.52 (1.09-1.98)

3.10 (2.38-3.86)

0.22 (0.07-0.51)

0.63 (0.341.06)

0.60 (0.39-0.83)

1.86 (1.33-2.53)

1.59(1.14-2.17)

3.19 (2.28434)

aOne value (1.63) excluded as outlier.

The effect of TPA, added at different time intervals, on the transformation

frequencies induced with Et0 is presented in Table II. The highest transformation

frequencies were observed when TPA was added to the medium directly after Et0

treatment as described in ‘Materials and Methods’. The effect of TPA diminished at

48 h and was not detectable when TPA was added at day 5.

Statistical treatment of data

The frequencies of transformed cells were adapted to a linear model (Y = a +

bD) using weighted linear regression, with weights proportional to numbers of sur-

viving cells in each assay. This model was preferred to the Poisson regression model

[19], since we observed that values in the TPA series were over-spread compared to

the Poisson assumption. The estimates of a (background frequency of transformed

cells) and b (slope of the dose-response curve) are presented in Table III.

The values and standard errors of the ratios of slopes (the rad-equivalence coeffi-

cients) are estimated to be 78 + 7 rad/mMh without TPA, and 75 + 26 rad/mMh

in the presence of TPA. The ratios between slopes in the presence and the absence

of TPA are 2.5 f 0.6 and 2.4 + 0.7 for gamma-irradiation and Et0 treatment, re-

spectively.

311

TABLE II

EFFECT OF 2.5 mMh Et0 ON THE TRANSFORMATION OF C3H/lOTl/2 CELLS IN THE PRES-

ENCE OF TPA, ADDED IMMEDIATELY, AT 48 h OR AT DAY 5

Treatment

Control

Control + TPA,

added immediately

Control + TPA,

added at 48 h

Control + TPA,

added at day 5

Et0

Et0 + TPA,

added immediately

Et0 + TPA,

added at 48 h

Et0 + TPA,

added at day 5

Total no.

of survivors

191340

34110

47200

47920 13128 0.27 (0.1550.48)

61720 31146 0.50 (0.314.71)

40500

31200

No foci/

No. flasks

141137

4123

13/30

12135

35130

24124

Mean value of transformation

frequency (foci per 10’ survivors,

with 95% confidence interval;

based on Poisson distribution)

0.07 (0.040.12)

0.12 (0.03-0.30)

0.28 (0.1550.48)

1.78 (1.39-2.24)

1.12(0.78-1.56)

0.78 (0.5G1.16)

DISCUSSION

The results presented in this paper demonstrate that TPA has an enhancing effect on the transforming abilities of Et0 and gamma-radiation and that the magnitude of the effect is similar for the two agents. Balcer-Kubiczek and Harrison [13] studied the effect of TPA on X-ray-induced transformation frequencies in C3H/lOT1/2 cells for different doses up to 400 rad. Their data suggest that the dose-response curve in the absence of TPA might be linear up to approximately 200 rad but markedly curvilinear above this dose, whereas in the presence of TPA the dose-response curve is linear in the dose range studied. The average transformation enhancement due to TPA was approximately 4 in the dose range O-200 rad. The initial slope of the dose- response curve in the absence of TPA at high dose rate (400 rad/min) was estimated to be 2.33 x 10V4 Gy-‘, i.e. 0.0233 x 10e4 radd’.

The data on gamma-radiation-induced transformation frequencies presented here are compatible with those of Balcer-Kubiczek and Harrison. The average transfor- mation enhancement due to TPA (2.5) is somewhat lower than the value presented by these authors and the slope of the dose-response curve in the absence of TPA (0.032 x 10S4 rad-‘) is somewhat higher, which, in fact, is expected since the esti- mates of TPA enhancement and slope are based on a linearization of the dose-re- sponse curve in the broader dose interval MOO rad.

312

TABLE III

ESTIMATES OF a AND b IN A LINEAR MODEL Y = a + bD FOR THE FREQUENCIES OF

TRANSFORMED CELLS

Treatment a k SE b k SE

(x 104) (x 104)

Et0 0.7 * 0.4 2.5 * 0.2

Et0 + TPA 1.7 f 4.6 6.0 + 1.6

Gamma-radiation 0.7 * 0.2 0.032 & 0.002

Gamma-radiation + TPA 0.7 & 3.3 0.080 + 0.018

Dose (D) is given in mMh (EtO) or rad (gamma-radiation)

In the previous paper [19] the rad-equivalence of Et0 in the absence of a tumor promoter was calculated to be 90.8 f 31.8 rad/mMh (95% confidence interval, CI). The values in this study, 78 + 14 rad/mMh (95% CI) in the absence of TPA and 75 f 52 (95% CI) in its presence are compatible with previous estimates and with the values for Et0 (4&200 rad/mMh) obtained in other experimental systems [2 11.

The present study shows that promotion by TPA had no influence on the rad- equivalence value of EtO. Additional studies on other genotoxic compounds in the presence and absence of a cancer promoter would be of interest in order to verify the generality of the rad-equivalence approach.

ACKNOWLEDGEMENTS

Thanks are due to Alan Wright (Shell Research Limited, Sittingbourne, England) for fruitful discussions, and Gian-Paolo Scalia-Tomba (Department of Mathematical Statistics, University of Stockholm) for help in the statistical evaluation of the data. The authors are grateful to Krystyna Hakansson for skilled technical assistance. This work was supported financially by the Swedish Natural Science Research Council, the Swedish Cancer Society, the Ivar Bendixsons Foundation (grant to A.K.), the Bank of Sweden Tercentenary Foundation and by Shell Internationale Research Maatschappij B.V., The Netherlands.

REFERENCES

Reznikoff, C.A., Brankow, D.W. and Heidelberger, C. (1973) Establishment and characterization of

a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer

Res. 33,323ll3238.

Bertram, J.S. and Heidelberger, C. (1974) Cell cycle dependency of oncogenic transformation induced

by N-methyl-N’-nitro-N-nitrosoguanidine in culture. Cancer Res. 34, 526-537.

313

3 Mondal, S. and Heidelberger, C. (1976) Transformation of C3H/lOT1/2 CL 8 mouse embryo fibro-

blasts by ultraviolet irradiation and phorbol ester. Nature 260, 71@711.

4 Terzaghi, M. and Little, J.B. (1976) X-radiation induced transformation in a C3H mouse embryo-de-

rived cell line. Cancer Res. 36, 136771374.

5 Kennedy, A., Cairns, J. and Little, J.B. (1984) Timing of the steps in transformation of C3H lOTlj2

cells by X-irradiation. Nature 307, 85-86.

6 Borek, C. (1985) Cellular and molecular mechanisms in malignant transformation of diploid rodent

and human cells by radiation. In: J.C. Barett and R.W. Tennant (Eds.), Carcinogenesis, Vol. 9. Raven

Press, New York, pp. 365-378.

7 Hill, C.K., Elkind, M.M. and Han, A. (1985) Role of repair processes in neoplastic transformation

induced by ionizing radiation in C3H/lOT1/2 cells. In: J.C. Barrett and R.W. Tennant (Eds.), Carcino-

genesis, Vol. 9. Raven Press, New York, pp. 379-397.

8 Bradshaw, T.K. (1986) Cell transformation: the role of oncogenes and growth factors. Mutagenesis

1,91-97.

9 Barrett, J.C. and Elmore, E. (1985) Comparison of carcinogenesis and mutagenesis of mammalian cells

in culture. In: W.G. Flamm and R.J. Lorentzen (Eds.), Mechanisms and Toxicity of Chemical Carcino-

gens and Mutagens, Vol. XII. Princeton Sci. Publ. pp. 171-206.

10 Kennedy, A.R., Fox, M., Murphy, G. and Little, J.B. (1980) Relationship between X-ray exposure

and malignant transformation in C3H lOT1/2 cells. Proc. Natl. Acad. Sci. USA 77, 726227266.

11 Elkind, M.M., Hill, C.K. and Han, A. (1985) Repair and misrepair in radiation-induced neoplastic

transformation. In: E. Huberman and S.H. Barr (Eds.), Carcinogenesis, Vol. 10. Raven Press, New

York, pp. 317-336.

12 Boreiko, C.J. (1985) Mechanistic aspects of initiation and promotion in C3H/lOT1/2 cells. In: J.C.

Barrett and R.W. Tennant (Eds.), Carcinogenesis, Vol. 9. Raven Press, New York, pp. 1533165.

13 Balcer-Kubiczek, E.K. and Harrison, G.H. (1988) Effect of X-ray dose protraction and a tumor pro-

moter on transformation induction in vitro. Int. J. Radiat. Biol. 54,81-89.

14 Frazelle, J.H., Abernethy, D.J. and Boreiko, C.J. (1984) Enhanced sensitivity of the C3H/lOT1/2 cell

transformation system to alkylating and chemotherapeutic agents by treatment with 120tetradecan-

oylphorbol-13-acetate. Environ. Mutagen. 6, 81-89.

15 Kennedy, A. (1985) Evidence that the first step leading to carcinogen-induced malignant transforma-

tion is a high frequency, common event. In: J.C. Barrett and R.W. Tennant (Eds.), Carcinogenesis,

Vol. 9. Raven Press, New York, pp. 355364.

16 Diamond, L. (1987) Tumor promoters and cell transformation. In: G. Grunberger and S. Goff (Eds.),

Mechanisms of Cellular Transformation by Carcinogenic Agents. Pergamon Press, Elmsford, NY, pp.

73-l 34.

17 Castagna, M., Takai, Y., Kaibuchi, K., Sane, K., Kikkawa, U. and Nishizuka, Y. (1982) Direct activa-

tion of calcium-activated phospholipid-dependent protein kinase by tumor-promoting phorbol esters.

J. Biol. Chem. 257,7847-7851.

18 Ehrenberg, L. (1980) Purposes and methods of comparing effects of radiation and chemicals. In: Ra-

diological Equivalents of Chemical Pollutants. IAEA, Vienna, pp. 23-36.

19 Kolman, A., Naslund, M., Osterman-Golkar, S., Scalia-Tomba, G.-P. and Meyer, A. (1989) Compara-

tive studies of in vitro transformation by ethylene oxide and gamma-radiation of C3H/lOT1/2 cells.

Mutagenesis 4, 5861.

20 Meyer, A., McGregor, D. and Styles, J. (1984) In vitro cell transformation assays. UKEMS Sub-com-

mittee on Guidelines for Mutagenicity Testing (Part II A), Ch. 6, pp. 123-135.

21 Kolman, A., Segerbbk, D. and Osterman-Golkar, S. (1988) Cancer risk estimation of genotoxic chem-

icals through the rad-equivalence approach. In: H. Bartsch, K. Hemminki and I.K. O’Neill (Eds.),

Methods for Detecting DNA Damaging Agents in Humans: Applications in Cancer Epidemiology and

Prevention. IARC (Lyon), 89, pp. 258-264.

22 Ehrenberg, L., Moustacchi, E. and Osterman-Golkar, S. (1983) Dosimetry of alkylating agents and

dose-response relationships of their effects. Mutat. Res. 123, 121-182.