The effect of complete carcinogens on intercellular transfer of lucifer yellow in fibroblast culture

Cell Biology and Toxicology, Vol. 6, No. 1, 1990 47

THE EFFECT OF COMPLETE CARCINOGENS ON INTERCELLULAR TRANSFER OF LUCIFER YELLOW

IN FIBROBLAST CULTURE

IRENE V. BUDUNOVA,* LEONID A. MITTELMAN,** AND GENNADY A. BELITSKY*

*Laboratory of Carcinogen Screening Methods All-Union Cancer Research Center of the AMS USSR

Moscow, USSR **Department of Mathematical Methods in Biology

Laboratory of Molecular Biology and Bioorganic Chemistry Moscow State University

Moscow, USSR

S U M M A R Y

The effect on permeability of gap junctions of complete powerful carcinogens, 3-methylcholanthrene (MC), 7,12-dimethylbenz(a)anthracene (DMBA), ethyl methanesulfonate (EMS), and weak carcinogens, benz(a)anthracene (BA), benzo(e)pyrene (B(e)P) as well as the arylhydrorylase inhibitor 7,8-benzoflavone (7,8-BF) has been studied with the use of a dye-coupling technique and transformed Djungarian hamster DM15 fibroblasts. MC, EMS and 7,8-BF were found to exert a strong inhibitory effect on cell-to-cell dye transfer. BA and DMBA had the uncoupling activity only in 2 out of 4 experiments. B(e)P was not shown to affect LY transfer between DM15 cells. The uncoupling effect of MC, 7,8-BF and EMS (only when EMS used at the concentration of 600 /zg/ml but not 1000 #g/ml) appeared reversible. The causes of failure to detect DMBA and B(e)P effects on gap junctions are discussed.

INTRODUCTION

The hypothesis of 2-stage carcinogenesis implies that complete carcinogens possess both initiating and promoting activities. Based on this assumption, complete carcinogens may be expected to induce in vivo and in vitro effects similar to those

1. Address correspondence to: I.V. Budunova, M.D., Laboratory of Carcinogen Screening Methods, All-Union Cancer Research Center of the AMS USSR, Moscow 115478, Kashirskoye shosse 24, USSR.

2. Key words: dye transfer, intercellular communication, complete carcinogens. 3. Abbreviations: B(a)P,benzo(a)pyrene; B(e)P,benzo(e)pyrene; BA, benz(a)anthracene; 7,8-BF, 7,8-

benzoflavone; DMBA,7,12,dimethylbenz(a)anthracene; MC,3-methylcholanthrene; EMS, ethyl methanesulfonate; LY, Lucifer Yellow; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; PAH, polycyclic aromatic hydrocarbons.

Cell Biology and Toxicology, Vol. 6, No. 1, pp. 47-62 Copyright © 1990 Princeton Scientific Publishing Co., Inc.

ISSN: 0742-2091

48 Budunova, et al

induced by classical promoters: phorbol esthers, organochlorine pesticides, etc. One of the most typical properties of tumor promoters is their capacity to inhibit intercellular communications (Trosko, 1987; Williams, 1987; Yamasaki, 1988). For the past decade over 100 tumor promoters have been studied in various assays assessing the functional state of gap junctions (Elmore et al., 1985; 1988; Jone et al., 1987; Malcolm et al., 1984; Noda et al., 1981; Telang et al., 1982; Trosko et al., 1982; Williams et al., 1981). The majority of the promoters tested have been shown to inhibit intercellular communiucation in vitro. Numerous attempts have been made to demonstrate the uncoupling effect of complete carcinogens including both direct (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4- nitroquinoline-N-oxide) and indirect (polycyclic aromatic hydrocarbons (PAH), aflatoxins) carcinogens, but the assays of metabolic cooperation inhibition or electrical coupling inhibition yielded no positive results (Enomoto et al., 1981; Malcolm amd Mills, 1984; Newbold and Amos, 1981; Noda et al., 1981; Telang et al., 1982; Jone et al., 1987). Thus, the effect of complete carcinogens on gap junctions still has not been elucidated.

In the present experiments, we assessed the effect of MC, DMBA, BA, B(e)P and 7,8-BF and of the direct acting carcinogen EMS on gap junction permeability using the method of intracellular dye-coupling. This assay allows the study of responses of individual cells to a carcinogen and, importantly, the changes in gap junction permeability at various time points can be followed. The target cells used in this study were SV-40 transformed Djungarian hamster fibroblast culture (DM 15 cell line), which were previously reported to be well-coupled and sensitive to the uncoupling effect of tumor promoters of varying organotropy (Budunova et al., 1989). DM 15 cells preserve a certain amount of microsomal monooxygenases, such that they are able to metabolize procarcinogens as evidenced by their sensitivity to the toxic effects of PAH and other procarcinogens (Kadyrova et al., 1986).

MATERIALS AND METHODS

Cell Culture. The experiments were performed on SV-40 transformed Djungarian hamster DM15 fibroblasts (obtained from Dr. E. S. Kakpakova, All-Union Cancer Research Center, Moscow). The cells were cultured at 37 ° in Eagle's MEM (Institute of Poliomyelitis and Virus Encephalitis, AMS USSR) supplemented with 10% BS (Moscow, USSR) and 0.1 mg/ml monomycin.

Test Chemicals. PAH: MC, DMBA, benzo(a)pyrene (B(a)P), B(e)P (Fluka AG), BA (Kodak) and 7,8-BF (Aldrich) were dissolved in acetone (VEB Laborchemie, Apolda, DDR) and afterwards, in BS. EMS (Serva) was dissolved in culture medium.

B(a)P Metabolism. The capacity of cells to metabolize B(a)P was estimated by measuring the reduction of the PAH in the culture medium in the course of


incubation. The cells were plated in Carrel flasks (3-5 flasks per group per each experiment). 24 h later the medium was changed for that containing the carcinogen (0.1 /~g/ml, 0.5 /~g/flask) and incubated with B(a)P for 24-72 h. Thereafter, the medium from each flask as well as the cell suspension collected from the flasks' bottom with trypsin, were placed in a separate tube. B(a)P was extracted with n-octane for 3 h at 40 ° and quantitated using a spectral luminescence assay at the temperature of liquid nitrogen on a fluorescence spectrophotometer "Hitachi- 850" by the procedure previously described (Khesina et al., 1979).

Dye Transfer Assay. The cells were plated on coverslips in Petri dishes and incubated in 5% CO2 atmosphere (the concentration of cells was 2 X 105 cells/ml). 24-48 h later the medium was changed for that with PAH or EMS, the final concentration of acetone in the medium being 0.2-0.3%. The cells were incubated with PAH or EMS for 5-24 h; thereafter, the coverslips with cells were washed with lactalbumine solution and placed into the chamber with a constant circular flow of lactalbumine solution (15 ml volume) mounted on the microscope stage of the luminescent microscope (Lumam, USSR). The saturated solution of Lucifer Yellow (LY) (Sigma) in distilled water was injected by ionophoresis into donor cells through a glass microelectrode (diameter of the tip, 0.5 tzm). The measurements were carried out at room temperature. The number of stained recipient cells was counted 1 min after the injection was started as described elsewhere (Budunova et al., 1989).

The assessment of the toxic effect of PAH and EMS. The cells were grown on coverslips in Petri dishes (concentration of plating 2 X 105 cells/ml). 24 h later the medium was changed for that supplemented with the agent tested. 72 h later the cells were fixed and stained. The relative changes in monolayer density were determined as compared to control. The density of monolayer was defined as the mean number of cells within the microscope field of view (7.5 X 104 #m2). From 20 to 50 fields of view were counted in each preparation.

RESULTS

The most important characteristic of cells employed in studies of procarcinogen effects is their metabolic competence. The capacity of DM15 cells to metabolize PAH was assessed by metabolism of B(a)P, a model carcinogenic PAH. DM15 cells preserved the capacity to oxidize B(a)P as evidenced by a decreasing concentration of B(a)P in the incubation medium, more marked with longer incubation from 24 to 72 h (Table 1). It should be noted that the rate of B(a)P metabolism by transformed DMI5 cells was high enough (30% B(a)P was metabolized for 24 h, and 100% for 72 h) and comparable to that inherent to normal embryonic murine, rat and hamster fibroblasts as shown by us previously (Andrianov et al., 1976).

50 Budunova, et al

T A B L E 1

M e t a b o l i s m o f B e n z o ( a ) p y r e n e in D M 1 5 Cell Cultures

Percentage of B(a)P metabolized over various time intervals

Expt 24 h 48 h 72 h

1 - - 67-t- 12 95-t- 1 2 31 ___ 8 - - 99.4 -t- 0.I

The DM15 cells are characterized by a high level of intercellular LY transfer and they preserve the capacity to metabolize procarcinogens. Accordingly, they are a suitable model for study of the effects of carcinogens on gap junction permeability. In the present experiments, the inhibition of intercellular LY transfer was studied for the following compounds: 3 complete strong carcinogens, DMBA MC and EMS and 2 weak carcinogens, BA, which is a pure initiator in vivo (Slaga and Nesnow, 1985) and B(e)P, a pure promoter in vivo (Slaga and Nesnow, 1985). The attempts at studying the effect of B(a)P on gap junctions using the assay of LY intercellular transfer have failed due to extremely intensive fluorescence of B(a)P per se.

Only MC of all the PAH carcinogens studied in our experiments proved to effectively inhibit gap junction permeability. It led to decreased LY intercellular transfer in 4 out of 4 experiments. The effect of MC on gap junction permeability was increased after its concentration was elevated from 1 to 10 #g/ml and the time of incubation was prolonged from 5 to 24 h (Figs. la and lb). In optimal conditions MC produced a 2-3-fold decrease in the intercellular LY transfer, the effect being comparable to that of tumor promoters butylhydroxytoluene and DDT on DM15 cells reported previously (Budunova et al., 1989).

The effects of DMBA, BA and B(e)P on gap junction permeability proved much weaker and unstable. Only in 2 out of 4 experiments DMBA and BA did exert the uncoupling effect, the number of stained cells being decreased by 30-40% (Table 2). The uncoupling effect of B(e)P was noted in 1 out of 3 experiments (Table 2). To summarize the results, DMBA and BA proved to be weak inhibitors of gap junction permeability whereas B(e)P did not affect it at all.

The direct-acting carcinogen EMS appeared to exert a marked uncoupling effect at 24 h-incubation starting with the concentration of 600 #g/ml (Fig. 2). This concentration is about that employed in experiments on EMS-induced mutations in somatic cells and gene amplification in DM15 cells (Kadyrova et al., 1986). The effect was enhanced when its concentration was raised to 1000 #g/ml (Fig. 2). The concentrations of 1, 10, 50 and 300 /~g/ml produced no effect on LY intercellular transfer. When the time of incubation was shortened to 5 h the concentrations of EMS within the range of 300-1000 #g/ml did not exert the uncoupling effect.


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FIGURE la. 3-Methylcholanthrene-induced inhibition of Lucifer Yellow transfer between DMI5 cells. - -O- - : Dye-coupled cells/injection. - - o - - : % control monolayer density. The data of separate experiments are presented. Each data point is the mean -t- standard deviation of 10-20 separate injections. 3-methylcholanthrene exposure time: 24 hours (dye-coupling assay); 72 hours (monolayer density).

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5 24 Postinjection Time (Hours)

FIGURE lb. The effect of a single concentration of 3-methylcholanthrene over time on Lucifer Yellow transfer between DM15 cells. - - O - - Dye-coupled cells/injection. The data of separate experiments are presented. Each data point is the mean _ standard deviation of 10-20 separate injections. 3-methylcholanthrene concentration ----- 10#g/ml.

52 Budunova, et al

TABLE 2 Effect of Polycyclic Aromatic Hydrocarbons on Dye Transfer Between DM15 Cells

No. of Dye-coupled Cells/Injection Concentration PAH (#g/ml)

Expt No PAH O (solvent) 1 5 10

DMBA 1 14.8 _+ 2.7 9.0 + 1.1" 2 12.4 --4-_ 2.6 11.7 -- 2.3 10.3 -4- 2.0 3 11.6 + 1.8 - 9.5 -4- 1.1 4 14.0 -4- 1.0 - 9.3 __. 1.I*

BA 1 11.8 -4- 2.7 15.8 ___ 2.1 10.8 ___ 1.6 2 12.6 ___ 2.1 - 9.5 -4- 1.6 3 12.3 -4- 0.9 - 6.4 _+ 1.5" 4 12.4 -4- 1.3 - 7.5 --- 1.4"*

B(e)P 1 11.6 -4- 2.0 8.0 -4- 0.9 2 13.3 -4- 1.2 8.2 ___ 0.8* 3 14.0 ___ 1.0 11.5 ___ 1.2

Each point is the mean (___ SD) of 10-20 separate injections. Time of exposure to P A H ---- 24 h. The significance of difference between the indices of treated and control cultures was evaluated by Student's test.

*The differences are significant, p > 0.99. **The differences are significant, p > 0.95.

In parallel to the uncoupling effect of carcinogens on gap junctions, we studied their toxic activity. Cell survival following the exposure to carcinogens was assessed by changes in the density of cell monolayer culture. We preferred this index to that of changes in cloning efficiency routinely used. We and many other investigators (Trosko et al., 1985; Elmore et al., 1988) claim that the toxic effect should be evaluated in the conditions similar to those in experiments on inhibition of gap junction permeability, i.e., monolayer but not sparce cell cultures should be used.

Of all the compounds tested only D M B A and EMS exerted a marked toxic effect on monolayer cultures of DM15 cells. D M B A induced a 2.5-fold decrease in monolayer density at the concentration of 1 #g /ml following 72 h incubation (Table 3). When used at the concentration of 10 #g/ml it induced 10-fold decrease as compared with control. EMS induced 3-fold decrease in monolayer density at the concentration of 600 /~g/ml and 10-fold decrease at the concentration of 1000 #g/ml following 72 h incubation (Fig. 2). It seems important to us that by the time the effect of D M B A or EMS on gap junctions was estimated (24 h incubation with the carcinogen) no appreciable changes in cellular morphology were noted.


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FIGURE 2. Ethyl methanesulfonate-induced inhibition of Lucifer Yellow transfer between DM15 cells. - -O- - : Dye-coupled cells/injection. - - e - - : % control monolayer density. The data of separate experiments are presented. Each data point is the mean ___ standard deviation of 10-20 separate injections. Ethyl methanesulfonate exposure time: 24 hours (dye-coupling assay); 72 hours (monolayer density).

So, when we compared the toxic and uncoupling action of carcinogens we could conclude that not any toxic compound induces decreasing permeability of gap junctions before there occur morphological changes of cells.

It seems important to know whether the procarcinogen or its metabolites possess inherent promoting activity. To answer this question, at least in terms of evaluating the inhibitory action on gap junction permeability, we attempted to assess the changes in the effect of MC on gap junctions during cell incubation with 7,8- BF, a well-known PAH-hydroxylase inhibitor (Diamond and Gelboin, 1969; Huberman and Sachs, 1974). Our preliminary experiments showed that 7,8-BF reduced the toxic effect of DMBA on DM15 ceils following their incubation with both PAHs. The protective action of 7,8-BF was most marked when DMBA and 7,8-BF wer used in the ratio of 1:8 (Table 3). Our results agree with those on the inhibitory effect of 7,8-BF on toxic and mutagenic activities of PAH in fetal cell cultures (Huberman and Sachs, 1974) and directly indicate that 7,8-BF also inhibits PAH metabolism in tansformed DMI5 ceils.

Unexpectedly, our experiments have shown that 7,8-BF per se appears to be an effective inhibitor of LY intercellular transfer (Fig. 3). Likewise MC, in optimal conditions (10 ~g/ml concentration, 24 h incubation) 7,8-BF induced 2-3-fold reduction in the number of stained cells without affecting the cell survival. The preliminary data show that the uncoupling effect of MC (10 ~g/ml) and 7,8-BF (10 ~tg/ml) on gap junction permeability was not additive.

54 Budunova, et al

TABLE 3 Effect of 7,8-Benzoflavone on Toxicity of 7,12-Dimethyl-benz(a)anthracene

in DM15 Cultured Cells

Monolayer Density (% to control) Treatment No of expt 1 2

Control 100 100 DMBA 1 #g/ml 44 32.5 7,8-BF 8 #g/ml 132 84.5 DMBA 1 #g/ml + 102 88

7,8-BF 8 #g/ml

The cells were treated either with DMBA alone, or with aryl hydroxylase inductor 7,8-BF, or simultaneously with DMBA and 7,8-BF for 72 h. The toxic effect of DMBA was assayed as described in the section "Materials and Methods."

MC, 7,8-BF and EMS (only at the concentration of 600 #g/m1 but not of 1000 ~g/ml) exerted a reversible effect on cell contacts. 24 h after the compounds were washed out the number of stained cells approached the control level (Table 4). The reversibility of action of these agents on LY transfer is a significant although indirect evidence of their specific effect on cell-to-cell contacts which is unrelated to physical destruction of gap junctions.

DISCUSSION

If we assume that all complete carcinogens possess inherent promoting activity, which is a logical conclusion of the hypothesis of 2-stage carcinogenesis, we could expect that 4 of the compounds tested in our experiments would prove to be inhibitors of LY intercellular transfer: complete carcinogens including MC, DMBA, EMS and the promoter B(e)P. However, according to our data only 2 out of these 4 compounds exhibited a marked effect on intercellular communication (Table 5).

EMS is a direct-acting carcinogen. The data on its promoting activity in experiments in vivo are not available to us, however, it has been reported that its close analog, methyl methanesulfonate may be an active promoter during urinary b~adder carcinogenesis; the promoting activity has been demonstrated for some other alkylating direct-acting carcinogens (MNNG, ethylnitrosourea) during skin carcinogenesis (Tudor et a1.,1984; O'Connell et al., 1987).

MC belongs to those carcinogenic PAH which have been directly demonstrated to have promoting activity in vivo; MC has been shown to be a promoter in hepatocarcinogenesis (Odashima, 1959). Interestingly, MC can induce skin morphological changes in mice similar to those recorded after the application of TPA, one of the most active skin promoters (Biirki and Bresnik, 1975).


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FIGURE 3. 7,8-Benzoflavone-induced inhibition of Lucifer Yellow transfer between DM15 cells. - -O- - : Dye-coupled cells/injection. - - o - - : % control monolayer density. The data of separate experiments are presented. Each data point is the mean -t- standard deviation of 10-20 separate injections. 7,8-benzoflavone exposure time: 24 hours (dye-coupling assay); 72 hours (monolayer density).

Our results showing a weak unstable inhibitory effect of DMBA on intercellular communication and the absence of any uncoupling activity of B(e)P generally agree with the negative evidence reported previously in relation to these compounds and B(a)P (Enomoto et al., 1981; Newbold and Amos, 1981; Noda et al., 1981; Telang et al., 1982; Malcolm and Mills, 1984). Consequently, a number of carcinogenic PAH do not affect gap junction permeability in vitro. These findings are discussed below with respect to the following points: 1) Detection the biological effects of PAH is possible following their metabolic activation; 2) The compounds may possess inherent promoter activity, however, they do not affect gap junction permeability.

As well known, PAH per se are not mutagenic or carcinogenic but require conversion by enzymes to reactive intermediates (Miller and Miller, 1976). Hence it seems likely that any promoting activity of procarcinogens would also be mediated via their active metabotites. The importance of metabolism has been demonstrated with regard to the effect of xenobiotics on intercellular communication in cell cultures for the following promoters: carbon tetrachloride, sodium cyclamate, phenol (S~ez et al., 1987; Malcolm and Mills, 1987). In this case, the failure to detect the effect of PAH and aflatoxins on intercellular communication in the assay of metabolic cooperation inhibition in V79 cell culture (Newbold and Amos, 1981; Noda et al., 1981; Malcolm and Mills, 1984; Jone et al., 1987) can be explained first of all, by the inability of V79 cells to activate these compounds (Huberman and Sachs, 1974; Newbold et al., 1977). Since the level of PAH metabolism in cells is determined

56 Budunova, et al

not so much by basal activity of arylhydroxylase as by its inducibility, one can assume that the level of cell oxidation of B(e)P and DMBA, weak inducers of arylhydroxylase, is lower than that of MC or B(a)P, strong inducers (Gelboin, 1967; Lewin and Conney, 1967; Nebert and Gelboin, 1968). Indeed, as shown by our preliminary data, the oxidation rate of B(e)P by DM15 cells was significantly lower than that of B(a)P. Thus it seems likely that the weak effect of DMBA and B(e)P on intercellular communication is related to insufficient metabolic activation of these compounds. However, it should be noted that the absence of the uncoupling effect of complete carcinogens cannot be explained only by the inability of cells to activate these compounds. Thus, B(a)P did not inhibit metabolic cooperation either in V79 cultured cells or in rat cultured hepatocytes (Telang et al., 1982), although the latter preserve high level of activity of microsomal enzymes and generally are considered optimal for the in vitro studies of procarcinogenic action (Williams et al., 1982). Besides, some direct-acting carcinogens which do not require metabolic activation such as MNNG did not inhibit metabolic cooperation and electric coupling either (Noda et al., 1981; Enomoto et al., 1981).

Tumor promotion is a complicated multistage process. Its major essence is to create the conditions for the predominant clonal expansion of initiated cells, and apparently, this can be achieved in different ways (Williams, 1984). One of the possibilities is disordered functioning of gap junctions between normal and initiated cells (Mehta et al., 1986; Trosko, 1987; Williams, 1987; Yamasaki et al., 1987). An alternative variant is the so-called promotion by cytotoxic mechanisms (Trosko et al., 1985). Indeed, the destruction of considerable numbers of cells, particularly, following

TABLE 4 Reversibility of the Effect Exerted by Polycyclic Aromatic Hydrocarbons

and Ethyl Methane Sulfonate on Dye Transfer Between DMI5 Cells

No of Dye-coupled Cells/Injection Test Chemicals Time Post-wash (h)

Doses Control 0 24

MC 10 #g/ml 13.4 ___ 1.1 4.1 _ 1.1 13.9 ± 1.8 7,8-BF 10 #g/ml 11.4 ± 1.5 3.9 ± 0.7 11.9 ± 1.8 EMS 600 #g/ml 16.9 ± 2.1 9.3 _ 1.7 20.2 ± 2.6 EMS 1000 #g/ml 16.9 q- 2.1 7.5 + 1.8 6.9 _ 0.9

The cells were incubated with MC, or 7,8-BF, or EMS for 24 h, washed 3 times with lactalbumine solution and incubated for varying times in fresh Eagle's medium with 10% bovine serum. The extent of dye transfer was measured immediately after exposure (time point 0) and then 24 h later (24 h post-wash).

The data of separate experiments are presented. Each point is the mean (± SD) of 10-20 separate injections.

Cell Biology and Toxicology, VoL 6, No. 1, 1990 57

exposure to highly toxic compounds (CC14, chloroform, 3,4,5,3,'4,'5'- hexabrombiphenyl, palytoxin, etc.) exerts a promoting effect in case of liver and skin carcinogenesis (BiJrzsiSnyi et al., 1984; Jone et al., 1987; Jensen et al., 1983). The promoting activity of complete carcinogens methyl methanesulfonate and MNNG is postulated to relate to their cytotoxic activity and subsequent compensatory hyperplasia (Tudor et al., 1984; O'Connell et al., 1987). Many PAH, and DMBA in particular, are carcinogens with a marked toxic effect (Biirki and Bresnik, 1975; Graffi and Bielka, 1959). The combination of the toxic activity of PAH which is selective with respect to normal cells with other effects including PAH induction of ornithine decarboxylase (O'Brien, 1977), changes in growth factor binding to cell surface receptors (Karenlampi et al., 1983; Weinstein et al., 1984) may create conditions sufficient for the promotion of initiated cells without inhibiting the intercellular communication.

The uncoupling effect induced by the anticarcinogen 7,8-BF (Gelboin et al., 1970) on gap junction permeability was unexpected. The capacity of this compound to inhibit metabolism of other PAH (Diamond and Gelboin, 1969; Huberman and Sachs, 1974) underlies the mechanism of its anticarcinogenic effect manifested following simultaneous application of 7,8-BF with carcinogenic PAH. 7,8-BF was previously shown to induce some in vitro effects similar to those induced by carcinogenic PAH, e.g., it changed growth factor binding to cell surface receptors (Karenlampi et al., 1983). We expect that if an experiment is performed according to the scheme of 2-stage carcinogenesis, i.e., the animals receive long-term treatment with 7,8-BF following the exposure to MC or another initiator, then the promoting activity of this compound would be detected.

TABLE 5 The Effect of Polycyclic Aromatic Hydrocarbons and Ethyl Methanesulfonate

on Dye Transfer in DM15 Cells

Agent Carcinogenicity

Inhibition of Intercellular

Communication

Reproducibility of Results (No. of expts, with the

effect/total No. of expts)

MC ++ DMBA ++ BA + (initiator)* B(e)P + (promoter)* 7,8-BF -- (in special cases

is an anticarcinogen**) EMS ++

++

++

++

4/4 2/4 2/4 1/3 3/3

3/3

*Slaga et al., 1975. **Gelboin et al., 1970.

58 Budunova, et al

Thus, for the first time we have demonstrated the inhibiting action of 3 compounds on intercellular communication: two complete carcinogens MC and EMS and of aryl hydroxylase inhibitor 7,8-BF. Further work is necessary to better understand the inhibiting effect of these compounds on intercellular communication and their differences from carcinogenic PAH inactive in terms of their effect on gap junction.

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