Radiat. Environ. Biophys. 19, 247-258 (1981)

Radiation and Environmental Biophysics © Springer-Verlag 1981

Effects of a Combined Treatment with X-Rays and Phenols on Preimplantation Mouse Embryos in Vitro W.-U. Miiller, C. Streffer, and N. Zamboglou Institut fiir Medizinische Strahlenphysik und Strahlenbiologie, Universit~itsklinikum Essen, Hufelandstrasse 55, D-4300 Essen 1, Federal Republic of Germany

Summary. Phenols are found everywhere in the environment. Therefore, the investigation of possible interactions between phenols and radiation is of some interest. The effects of a combination of X-rays and phenols (phenol itself and p-nitrophenol) were measured by the preimplantation mouse embryo-system in vitro. The microscopic visible development up to 144 h post conceptionem (h.p.c.), the number of cell nuclei, the DNA-content of each nucleus, the mitotic index, the labelling index, and the number of micronuclei were determined. There was not any indication that the effect of the irradiation was enhanced in a synergistic manner by the presence of phenols. All parameters measured lead to the conclusion that the effects of phenols plus X-rays are, at most, additive.

Introduction Phenols represent a group of toxic substances found everywhere in the environment. They occur in water (in 1975 Rhine water near Karlsruhe contained 1 - 5 mg phenols/m3 water [24]), in the atmosphere (in 1973 in Cologne an average phenol content of 0.02-0.03 mg/m3 air was measured [2]), in soil, in food (either naturally or added as antioxidants, e.g., butylated hydroxytoluene), and in cigarettes (the smoke from 100 cigarettes contains about 10 mg phenol and 50 mg catechol besides other phenols [36]). The content of phenols in air, water, and soil mainly originates from industry (especially coal manufacturing and certain plastic material producing industries), domestic heating (an oil oven can release up to 80 mg phenol/kg oil [14]) and exhaust gas from cars (between 0.6-28 mg phenol/1 fuel depending on the type of fuel [6]). Furthermore, one must keep in mind the various possibilities of metabolic breakdown of complex molecules (e.g., protein degradation) to 0301-634X/81/0019/0247/$ 02.40

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phenols. An interesting example in this connexion is the degradation of parathion - an organophosphate insecticide - to p-nitrophenol [9, 25]. An interaction between phenol and X-rays is not improbable because phenol is a promoter in carcinogenesis [4, 27] and a mutagen in some test systems (for a review see [8]). Chromosome fragmentation in cells of root tips of Allium cepa, however, was only rarely observed after phenol application [19]. p-Nitrophenol, on the other hand, does not show promoting properties [4] nor does it induce mutations in Salmonella [20]; but it was found to cause chromosome fragmentation in root tips of Allium cepa [30]. Both, phenol and p-nitrophenol, exert some influence on repair processes [26]. The concentrations needed, however, are rather high (10-100 mM for phenol and 1 mM for p-nitrophenol); additionally, semiconservative DNA-replication is inhibited at these concentrations, too. According to Morimoto et al. (1976) phenol inhibits repair of radiation-induced chromosome breaks in cultured human leukocytes. From the above mentioned facts it is clear that the investigation of a possible interaction of phenols and X-rays in organisms is of great interest. Therefore, the effects of a combined treatment of phenols (phenol itself and p-nitrophenol) with X-rays were examined. Test-objects were preimplantation mouse embryos in vitro [34], the radiosensitivity of which was already shown by several authors (e.g., [1, 11, 12, 16, 22, 321).

Materials and Methods

Chemicals Phenol was obtained from Merck (Darmstadt), p-nitrophenol from Calbiochem (Gief3en), and silicone oil (Tegiloxan 50) from Goldschmidt (Essen).

Isolation and Culture of Embryos One male and three female mice (not superovulated!) of an inbred strain (Radiologisches Institut, Freiburg i. Br.) were mated for 3 h. Those females with a vaginal plug were killed about 30 h later and their oviducts were flushed with culture medium BMOC-2 [5]. The embryos (all of them in the two-cell-stage at this time) were collected in i ml medium in a watch glass and subsequently distributed into plastic dishes (35 x 10 ram, Greiner, Ntirtingen) containing 2 ml medium with the appropriate concentration of phenols. Culturing was performed at 37° C and 10% CO2. After 24 h the embryos were washed with phenol-free medium and transferred to 0.1 ml phenol-free medium overlayed with 3.5 ml silicone oil. This procedure is necessary because phenol is readily soluble in silicone oil; therefore, it is not possible to culture the embryos in a phenol-containing medium under silicone oil.

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X-Irradiation The embryos were irradiated in the late G2-phase (determined by DNA-fluorescence; see [34]) of the two-cell-stage (32 h p.c.) with the stabilipan X-ray machine (Siemens, Erlangen) at 15 mA, 240 kV, 0.5 mm copper filter, and a dose rate of 1 Gy/min. Irradiation of embryos was performed in medium and at room temperature.

Determination of Parameters a) Cell Number and Mitotic Index. In embryos containing up to eight cells the cell boundaries are clearly recognizable under the microscope. From the morula stage onwards (72 h p.c.) the embryos were spread on a glass slide after treatment with 1% sodium citrate solution and application of fixative solution (acetic acid/ethanol 1 : 3 [35]). The nuclei were stained with Mayers Hfimalaun and counted. The number of nuclei in mitosis was also determined. b) Labelling Index. The determination of the labelling index was performed by application of 3H-thymidine (2 ~tCi/ml; spec. act. 48 Ci/mMole) for 15 rain immediately before termination of the culture. After washing the embryos three times in medium, they were spread on a glass slide as described in the previous section. The slides were dipped into gel emulsion (Ilford K2) and exposed for 3 weeks. After staining with Mayers Hfimalaun the labelled nuclei were counted. c) Relative DNA Content. The relative DNA content of the individual nuclei was determined with a microscope photometer (MPV 1, Leitz, Wetzlar) after staining the nuclei (prepared according section a) with ethidium bromide [18, 34] and measuring the fluorescence at 530 nm. d) Micronuclei. All particles possessing the following properties were counted as micronuclei: 1. Stainable with ethidium bromide. 2. Diameter between 1/10 and 1/5 that of the main nucleus. 3. Round-shaped with distinct boundaries. Results

1. Toxicity of Phenols The toxicity of phenols was measured by their effects on the microscopic visible development of the embryos. The number of cells per embryo (48 h p.c.) the formation of morulae (72 h p.c.), the formation of blastocysts (96 h p.c.), and the percentage of hatched blastocysts (144 h p.c.) was determined. Figure 1 shows the results for phenol and p-nitrophenol. Up to 10 -4 M phenol the development of the embryos is only slightly if at all impaired. 48 h p.c. concentrations between 0.5 × 10-3 M and 0.5 × 10 -2 M

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Fig. 1. Effects of different phenol (© O) and p-nitrophenol (x x ) concentrations on the development of preimplantation mouse embryos in vitro. Variables determined (control value): 48 h p.c.: Average cell number/embryo (5.80); 72 h p.c.: Formation of morulae/culture (84%); 96 h p.c.: Formation of blastocysts/culture (90%); 144 h p.c.: Hatched blastocysts/culture (83%). Arrows indicate the concentrations used in combination experiments Table 1. Effects of a combined treatment with phenols and X-rays on the development of preimplantation mouse embryos. (Variables determined: 48 h p.c. and 56 h p.c. = average cell number/embryo; 72 h p.c. = formation of morulae/culture; 96 h p.c. = formation of blastocysts/culture; 144 h p.c. = hatched blastocysts/culture) Treatment

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seem to damage the embryos to a very similar extent; but if the development until 144 h p.c. is followed, it can be seen that the embryos, which were grown for 24 h in 0.5 × 10 -3 M phenol, are able to recover, whereas no further development occurs after 0.5 × 10 -2 M. 0.5 mM phenol was chosen for the combination experiments with X-rays. On average p-nitrophenol seems to be about ten times more toxic than phenol. A concentration of 10 -6 M p-nitrophenol does not influence the development of the embryos. 10 -5 M, however, slightly impairs the first part of development (i.e., until 96 h p.c.), but the embryos are able to return to the control value at 144 h p.c. 0.5 × 10 .5 M has similar effects to 10 .5 M, but there is no recovery at the end of the culture; this concentration was used for the experiments combined with X-rays.

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2. Effects by X-Rays The relation between the dose of X-rays and the effect on the proliferation of mouse embryos after irradiation in the late G2-phase of the two-cell-stage is not linear: Up to 0.24 Gy there is no difference in comparison to the control values [22]. After a radiation dose of 0.94 Gy the cell numbers are 2 0 - 3 0 % lower than the corresponding control values (Tables 2 and 3). This radiation dose was used in the combination experiments.

3. Effects of a Combination of Phenols and X-Rays Immediately (within 2 - 5 rain) after irradiation the embryos were transferred to 2 ml medium with or without phenol. At first the influence of the combination of phenols and X-rays on the microscopic visible development was determined. Phenol (0.5 raM), p-nitrophenol (0.05 mM), and X-rays (0.94 Gy), if applicated singly, lead to an inhibition of development between 15 and 30% compared to control embryos. If the single effects, which phenols and X-rays have alone, are added, it can be seen by comparing this calculated - expected - value with the measured value after combined treatment that both results are nearly identical (Table 1). Thus, the extent of the damage which is produced by a combined application of phenols and X-rays corresponds to the extent of damage obtained if the effects of the single treatments are added to each other. An additive effect is also observed for a combination of 0.94 Gy X-rays and 0.1 mM phenol, a concentration which does not impair the development as can be seen from Fig. 1. The microscopic visible development gives a first good hint for the effects of damaging influences. A much better quantitative measure is the number of nuclei formed during the culture of an embryo. Tables 2 and 3 show the changes in the number of nuclei after the different types of treatment; moreover, the tables present the influence of the agents on the labelling index and the mitotic index. The conclusion which was drawn from the results of the microscopic visible development is confirmed by the measurement of the number of nuclei (Tables 2 and 3): The numbers of nuclei calculated by addition of the single effects are either as high as the numbers of nuclei measured after the combined treatment or a little lower. The extent of damage is certainly not greater than would be expected from the addition of the single effects. Labelling index and mitotic index of the embryos treated are somewhat lower than in the control embryos at the beginning of the culture (until 96 h p.c.) and a little higher at the end of the culture period (from 120 h p.c. onwards). The impaired formation of cell nuclei after treatment with X-rays and phenols indicates that there is some influence of the agents on the cell cycle. Therefore, the distribution of nuclei within the cell cycle was determined with the aid of a microscope photometer (MPV) (Fig. 2 and Fig. 3). 72 h p.c. (the time

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of measurement) the embryos proliferate rapidly; the cell cycle time is about 1 2 - 1 3 h and the S-phase lasts about 6 - 8 h [34]. Consequently it is not surprising that most of the cell nuclei are in S-phase. The differences between the percentage of S-phase cells (determined by the MPV) and the extent of thymidine incorporating nuclei (measured by autoradiography) are not significant for the controls and for the combined treatment with X-rays and p-nitrophenol. The reason for the difference between the values for S-phase cells and for thymidine incorporating nuclei after the other treatments is not clear at present, but the problem is under investigation. There is no statistical significant difference in the cell cycle distribution after the different treatments except for the extent of hypoploidic and hyperploidic cell nuclei. Both phenol and p-nitrophenol lead to the formation of hyperploidic nuclei, a p h e n o m e n o n known for phenols [10]. X-rays on the other hand have a clear tendency to induce hypoploidic nuclei [22]. After the combined treatment

Combined Treatment with X-Rays and Phenols

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of phenols and X-rays hypoploidic as well as hyperploidic cell nuclei appear. A very sensitive measure for damage especially of the cell nucleus is the number of so-called micronuclei. Micronuclei are chromatin fragments of the cell nucleus which appear after treatment with ionizing radiation or chemicals [7, 15, 28]. Micronuclei can derive from acentric chromosome fragments which cannot be distributed among the daughter nuclei during mitosis, but which remain within cytoplasm [13]. Additionally, they can originate from di- or multicentrics connected by bridges or from a malfunction of the spindle apparatus [29]. Phenol and p-nitrophenol induce the formation of micronuclei; but as can be seen from Fig. 4, there is only a distinct formation of micronuclei if the proliferation of the embryos is strongly impaired by the toxicity of the phenols. In contrast, X-rays induce micronudei even if the proliferation is not greatly impaired. If the proliferation is inhibited in such a way that 25% less cell nuclei are formed in comparison to the control, phenol-treated embryos show 5.8 micronuclei per 100 main nuclei, p-nitrophenol-treated 4.7, but irradiated embryos 11. This may point to different mechanisms of the induction of micronuclei by phenols and X-rays as well as to a relation of these effects to cell proliferation. Though phenol and p-nitrophenol themselves induce the formation of micronuclei (Fig. 4), the number of micronuclei after a combined treatment with X-rays and phenols does not exceed the expected additive values: After combination of X-rays (0.94 Gy) and phenol (0.5 mM) 10.1 micronuclei per 100 cell nuclei were found 72 h p.c.; from the addition of the effects of the single agents a value of 11.6 micronuclei per 100 cell nuclei had to be expected. X-rays

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(0.94 Gy) plus p-nitrophenol (0.05 mM) delivered 10.5 micronuctei per 100 cell nuclei; the expected value was 11.9 micronuclei per 100 cell nuclei. Thus, the effects are by no means more than additive in the combined treatment.

Discussion Phenol and especially p-nitrophenol proved to be rather toxic to preimplantation mouse embryos in vitro. All variables measured - the microscopic visible development, the number of nuclei, the labelling index, the mitotic index, the distribution of nuclei within the cell cycle, the number of micronuclei - were influenced by the phenols in a dose-dependent manner after exceeding a certain threshold level. It is well known for a number of substances that they are able to modify the effects of ionizing radiation either in a synergistic or in an antagonistic way [23]. Because of the wide distribution of phenols in the human environment it is of great importance to investigate a possible interaction between phenols and radiation. The decision as to whether an effect is antagonistic, additive or synergistic is not as simple as one might expect [3, 31]. This depends on the fact that dose-effect curves are linear only in very rare cases. If, however, there is a deviation from linearity and nothing is known about the mechanisms of both agents it is not permissible merely to add the effects of the single agents and to infer antagonism, additivity or synergism from the comparison with the value observed after combined treatment. If the mechanisms are independent and without interaction, addition of the single effects may be correct; but if the mechanisms are similar and/or interaction occurs one must consider that the second agent raises the "concentration" of the first one, so that one would have to add doses rather than the quantitative response [17]. The isobologram-analysis described by Steel and Peckham (1979) is necessary whenever the detection of synergistic or antagonistic effects is claimed and at least one of the dose-effect curves deviates from linearity. If the addition of the single effects leads to the conclusion of mere additivity, an isobologram-analysis is not necessary, because the results must lie within the envelope of additivity in any case, as the addition of effects corresponds to the mode-I calculation performed by Steel and Peckham. Most of the data obtained after combination of phenol and p-nitrophenol with X-rays are explainable by an additive behaviour of phenols and X-rays. Only some results seemed to tend to a slight sub-additivity; the examination according the instructions of Berenbaum (1977) demonstrates the existence of a small tendency in the direction of subadditivity, but this tendency was not very marked. The problem of additivity is much simpler in the case of micronuclei: After irradiation the dose-effect curve is definitely linear under these conditions [21], and likewise after treatment with phenol and probably with p-nitrophenol, too, a linear dose-effect relationship is observed. Therefore the addition of effects is permissible independent of the mechanisms by which X-rays and

Combined Treatment with X-Rays and Phenols

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phenols act. The evaluation of this parameter also leads to the conclusion that the combined application of phenols and X-rays results in additive to subadditive effects under these experimental conditions. The results following a combined treatment by phenols and X-rays are different from those results which were detected with the same experimental system, where actinomycin D or lead chloride were combined with ionizing rays [33]. The combined effects of actinomycin D and tritiated water on the development of the embryos are higher than additive. Lead on the other hand, if combined with X-rays, shows only additive effects on the proliferation of the embryos, but a more than additive effect upon the cytogenetic level, i.e., on the formation of micronuclei. None of the parameters measured after the combined treatment of mouse embryos with phenol or p-nitrophenol and X-rays gives any indication for a synergism or a more than additive extent of damage; all data lie within the "envelope of additivity" or in the direction of a slight sub-additivity.

Acknowledgements. We wish to thank Ms. C. Fischer and Mrs. U. Kaiser for their valuable assistance. This work was supported by the Bundesministerium des fnnern of the Federal Republic of Germany.

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