PLEASE NOTE THAT TO OBTAIN DETAILS OF THE PROTOCOL YOU SHOULD REGISTER

TO OBTAIN DETAILED PROTOCOL YOU SHOULD FIRST REGISTER AT ECVAM SIS

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

Protocol no. 65
LUCIFER YELLOW INTERCELLULAR EXCHANGE ASSAY FOR TUMOUR PROMOTERS

The effect of the test substance on the transfer of the dye lucifer yellow between SV-40-transformed hamster fibroblasts is an indication of potential tumour-promoting activity.

CONTACT

Dr. Irina V. Budunova
Cancer Research Centre Russian AMN
Kashirskoe Shosse 24 115478 Moscow Russia
Tel: 7 (095) 323 58 22

RATIONALE

Disordered functioning of gap junctions between normal and initiated cells has been proposed as one possible mechanism of tumour promotion. Gap junction permeability may be evaluated directly by evaluating the intercellular transfer of a microinjected fluorescent dye.

BASIC PROCEDURE

SV-40-transformed Djungarian hamster fibroblasts are cultured on coverslips in the presence or absence of test compound. A solution of lucifer yellow is then injected into some cells by means of a glass microelectrode. The number of stained cells is counted 1 minute after the start of the injection.

CRITICAL ASSESSMENT

Although the multi-stage theory of carcinogenesis is generally accepted, most carcinogenesis screening targets only the identification of compounds capable of causing the initial genetic damage. Compounds that are active as tumour promoters at later stages of the process do not directly damage DNA at biologically active concentrations. For this reason, standard short-term carcinogenicity assays are not appropriate for the detection of tumour-promoting activity. The lack of knowledge about the various mechanisms involved in tumour promotion and the interrelationships that exist between them has hampered the development of assays for tumour promoters. In the present state of knowledge, the class of assays that appear to be most specific and valid from a theoretical viewpoint is the one which focuses on the effects of tumour promoters on intercellular communication (Budunova and Mittelman, 1991; Trosko et al, 1988; Yamasaki, 1990). Gap junctions are organelles which permit the exchange of small water-soluble molecules between adjacent cells. It has been suggested that tumour promoters may act by inhibiting intercellular communication via gap junctions, so as to isolate initiated cells from the restraining effects of adjacent normal cells. Since the first demonstrations in 1979 (Murray and Fitzgerald, 1979; Yotti et al, 1979) that the phorbol ester TPA, a tumour promoter, inhibits metabolic cooperation between mammalian cells, a number of studies have shown a qualitative correlation between in vivo promoting activity and the inhibition of intercellular communication in vitro as assessed by various assays of the functional state of gap junctions (e.g. Trosko et al, 1982; Telang et al 1982; Elmore et al, 1985, 1988). In addition to using tests of metabolic cooperation, another promising way to study the functional state of gap junctions is to evaluate the intercellular transfer of microinjected fluorescent dyes (Loewenstein, 1979). This method has a number of advantages over tests of metabolic cooperation. Only a small quantity of cells is required, and the results are rapidly obtained (1-2 days). It is possible to follow the responses of individual cells to the test substance at various time points. Furthermore, in contrast to tests of metabolic cooperation, the fluorescent method is a direct measure of gap junction permeability which does not rely upon the occurrence of any other process. The dye can be introduced into the cells in various ways (Budunova and Mittelman, 1991), including the FRAP method (fluorescence redistribution after photobleaching between cells prelabelled with a fluorescein derivative), microinjection through the wounded surface of the cell, or, as described in this protocol, microinjection via a microelectrode. The cells used in the assay, as described in this protocol, are SV-40-transformed Djungarian hamster DM15 fibroblasts. This cell line preserves microsomal monooxygenases to a certain degree, and so is capable of metabolizing procarcinogens (Kadyrova et al, 1986, Budunova et al, 1990). This is an important advantage, since it is known that many carcinogens require metabolic activation. DM15 cells are well-coupled under normal conditions (Budunova et al, 1986) and have been shown to be sensitive in this assay to the uncoupling effect of promoters that target various organs (Budunova et al, 1989). Fluorescent dye transfer has also been used to study the uncoupling effects of skin tumour promoters, phorbol esters and mezerein on mouse HEL-37 epidermal cells, BALB/c 3T3 fibroblasts, and human intestine epithelial cells (Friedman and Steinberg, 1982; Fitzgerald et al, 1983; Yamasaki et al, 1984), and of other types of promoters on V79 cells (Zeilmaker and Yamasaki, 1986). In a study of the effects of 8 promoters in the lucifer yellow assay (Budunova et al, 1989), 7 inhibited the transfer of the dye. 4 Promoters of skin carcinogenesis, 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein, A23187 and teleocidin, caused a strong inhibition (3-6-fold decrease in the number of stained cells) which was evident within 30 minutes. When the incubation period was extended to 20-24 hours, uncoupling no longer occurred with TPA, mezerein and teleocidin. The Ca-ionophore A23187 uncoupled cells in a narrow concentration range after a short exposure, and was lethally cytotoxic after a 24-hr exposure. Anthralin had a weak inhibitory effect. The liver cell promoters DDT and phenobarbital (PB) had opposing effects, with DDT exerting strong uncoupling activity which became more marked when the incubation time was lengthened from 4 to 48 hr, while PB enhanced the transfer of lucifer yellow over short periods of incubation and had no effect after prolonged incubation. Butylated hydroxytoluene was an effective uncoupler, with an optimal effect after 14 hr. The putative promoter sodium nitrite inhibited dye transfer, not by diminishing the number of cases of intercellular transfer, as was the case with most promoters studied, but by decreasing the number of stained recipient cells. The uncoupling effect was reversible in all the cases studied. In another study of complete carcinogens (Budunova et al, 1990), strong uncoupling activity was exhibited by 3-methylcholanthrene, 7,8-benzoflavone, and ethyl methanesulfonate (EMS). The uncoupling effect of the first two compounds was reversible, as was that of EMS at 600 but not at 1000 mg/ml. 7,12-Dimethylbenzanthracene and benz(a)anthracene gave inconsistent results, showing uncoupling activity in some experiments and not in others, while benzo(e)pyrene did not affect intercellular transfer of lucifer yellow. Budunova and Mittelman (1991) have reviewed results reported for about 30 compounds in the dye transfer assay. Good reproducibility was obtained between different laboratories in most cases. The temporary inhibition that occurs, for example, with mezerein, is typical of promoters which are activators of protein kinase C. After about 24 hours, the cells develop resistance to the effects of these compounds, and gap junction permeability returns to normal levels. DDT is representative of another class of promoters whose inhibitory effect increases with increasing exposure time. Phenobarbital has been suggested to have a 2-phase effect, increasing gap junction permeability over the first 4-5 hours, and causing a decrease in permeability in cells subjected to a 4-day exposure. The above results indicate that assay conditions, in particular incubation time and test substance concentration, need to be optimized in relation to the substance being tested. For this reason, three incubation times (1, 4 and 24 hr) are used in the protocol. It is also recommended to test at least three dilutions, starting from the concentration that causes morphological changes in 50% of the cells. When evaluating results, it is important to have an estimate for the toxicity of the test compound. A simple way of obtaining this is to determine the relative changes in monolayer density compared to the density of untreated control cultures. Decreased gap junction permeability that arises from cytotoxic effects will not occur at concentrations of the test compound that are insufficient to cause morphological changes in the cells. Demonstration of the reversibility of gap junction inhibition after removal of the promoter is a further method by which the specificity of the effect can be demonstrated. It is important to note that the assay provides only a qualitative indication of potential tumour-promoting capability. The degree of promoting activity shown by a substance in vivo cannot be predicted from the degree of its uncoupling effect in vitro. For example, it has been found (Budunova et al., 1989) that weak promoters such as mezerein and A23187 had a marked uncoupling effect in vitro which equalled that seen with strong promoters such as TPA and DDT. In conclusion, the lucifer yellow assay is a rapid, direct and relatively reproducible method for the identification of tumour promoters. It may be useful as a tool to investigate the process of carcinogenesis, and should also be considered for inclusion in a battery of methods for promoter screening.

CHEMICALS TESTED

12-O-Tetradecanoylphorbol-13-acetate Mezerein A23187 Teleocidin Anthralin DDT Phenobarbital Butylated hydroxytoluene Sodium nitrite 3-methylcholanthrene 7,8-Benzoflavone Ethyl methanesulfonate 7,12-Dimethylbenzanthracene Benz(a)anthracene Benzo(e)pyrene Asbestos Nigericin Tobacco smoke condensate

TEST STATUS

The dye microinjection assay for tumour promoters has been widely accepted for use by laboratories worldwide. Results from this assay are incorporated into data for evaluation of carcinogenicity in the IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans.

REFERENCES

  1. Budunova, I.V., Mittelman, L.A., Belitsky, G.A., and Chailakhjan, L.M. (1986) Inhibitory effect of tumor promoters on the Lucifer Yellow intercellular exchange in culture of transformed Djungarian hamster fibroblasts. Doklady Acad. Sci. USSR 290: 1334-1338.
  2. Budunova, I.V., Mittelman, L.A., and Belitsky, G.A. (1989) Identification of tumor promoters by their inhibitory effect on intercellular transfer of lucifer yellow. Cell biology and toxicology 5: 77-89.
  3. Budunova, I.V., Mittelman, L.A., and Belitsky, G.A. (1990) The effect of complete carcinogens on intercellular transfer of lucifer yellow in fibroblast culture. Cell biology and toxicology 6: 47-61.
  4. Budunova, I.V., and Mittelman, L.A. (1991) Inhibition of intercellular communication and tumour promoter screening. in Intercellular communication (ed. F. Bukauskas) Manchester; Manchester University Press 109-120.
  5. Budunova, I.V., and Mittelman, L.A. (1992) The effect of K+/H+ antiporter nigericin on gap junction permeability. Cell biology and toxicology 8: 63-78.
  6. Elmore, E., Korytynski, E.A., and Smith, M.P. (1985) Tests with Chinese hamster V79 inhibition of metabolic cooperation assay. in: Progress in mutation research, Vol. 5. (J. Ashby, F.J. de Serres et al, eds.). Amsterdam; WHO/Elsevier. p. 597-612.
  7. Fitzgerald, D.J., Knowles, S.E., Ballard, F.J., and Murray, A.W. (1983) Rapid and reversible inhibition of junctional communication by tumor promoters. Cancer Res. 43: 3614-3618.
  8. Friedman, E.A., and Steinberg, M. (1982) Disrupted communication between late-stage premalignant human colon epithelial cells by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 42: 5096-5105.
  9. Kadyrova, E.L., and Kopnin, B.P. (1986) Induction of gene amplification in Djungarian hamster cells by some carcinogens. Bulletin of experimental biology and medicine 101: 749-752 (in Russian).
  10. Loewenstein, W.R. (1979) Junctional intercellular communication and the control of growth. Biochem. Biophys Acta 560: 1-65.
  11. Murray, A.W., and Fitzgerald, D.J. (1979) Tumor promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem. Biophys. Res. Commun. 91: 395-401.
  12. Telang, S., Tong, C., and Williams, G.M. (1982) Epigenetic membrane effects of a possible tumour promoting type on cultured liver cells by the nongenotoxic organochlorine pesticides chlordane and heptachlor. Carcinogenesis 3: 1175-1178.
  13. Trosko, J.E., Yotti, L.P., Warren, S.T., Tsushimoto, G, and Chang, C.C. (1982) Inhibition of cell-cell communication by tumour promoters. in: Carcinogenesis V.7, Cocarcinogenesis and biological effects of tumor promoters. (E. Hecker, N.E. Fusenig, W. Kunz, F. Marks, and H.W. Thielman, eds.). New York; Raven Press. p. 565-585.
  14. Trosko, J.E., Chang, C.C., Madhukar, B.V., Oh, S.Y., Bombick, D., and El-Fouly, M.H. (1988) Modulation of gap junctional intercellular communication by tumor promoting chemicals, oncogenes and growth factors during carcinogenesis. in: Gap junctions (E.L. Herzberg and R.G. Johnson, eds.). New York; Alan Liss. p. 435-448.
  15. Yamasaki, H. (1984) Modulation of cell differentiation by tumor promoters. in: Mechanisms of tumor promotion. V.4, Cellular responses to tumor promoters. (T.J. Slaga, ed.). Boca Raton; CRC Press. p. 1-26. Yamasaki, H. (1990) Gap junctional intercellular communication and carcinogenesis. CArcinogenesis 11: 1051-1058.
  16. Yotti, L.P., Chang, C.C., and Trosko, J.E. (1979) Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science 206: 1089-1091.
  17. Zeilmaker, M.J., and Yamasaki, H. (1986) Inhibition of junctional intercellular communication as a possible short-term test to detect tumor-promoting agents: result with nine C chemicals tested by dye transfer assay in Chinese hamster V79 cells. Cancer Res. 46: 6180-6186.

IP-65 © December, 1992