Studies of the rad-equivalence of ethylene oxide in the presence and absence of...
Transcript of Studies of the rad-equivalence of ethylene oxide in the presence and absence of...
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)
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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).
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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.
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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.
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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.
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