Exposure to BSDR Conversion BSDR to Effect Conversion
Host Factors Environmental
Conditions Intra(Inter)- Subject Variability Direct Empirical (D.E.) Semi-Empirical Extrapolation (S.E.E.) Empirical Correlation (E.C.) Theory-based Inference (T.B.I.) Existential Insight (E.I.) 1 Overall Assessment
the target context of in-vivo carcinogenicity are valid. For in-vitro
nneasurement of GJIC, the broad category of "environmental conditions" will be discussed, in the form of the following list.
Assumptions and Antecedent Conditions for Inter-Context Extrapolation from Observation of GTIC Down-Regulation to In-Vivo Carcinogenicity
1. Heterologous Cell System. As it is the growth of iiutiated cells which is etiologically related to the production of tumors, experiments should
evaluate communication between "normal" cells and initiated or
preneoplastic cells of the same tissue or a biologically relevant, contiguous
tissue.
2. Cell Types. Established cell lines, such as C3H/10T1/2 and BALB/c 3T3 cells have well known growth characteristics and are easily grown to large cell numbers for these experiments. However, attention must be paid to the passage number of immortalized cells used to ensure inter-experiment comparability, as phenotypic or genotypic changes may occur.
Terzaghi-Howe (1987) found that, unlike primary epithelial cells,
immortalized tracheal epithelial cells were imable to suppress the growth of initiated cells in reconstructed rat trachea. The process of immortali2ing cells can have varying effects on their GJIC competence, frequently causing lower levels of homologous and heterologous communication. This may mean
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that established cell lines would be more sensitive to down-regulation by environmental substances being tested (ie, exhibit a lower threshold of GJIC regulating effects), thus providing a more conservative risk characterization. However, this possible advantage would seem to be outweighed by the fact that immortalization often produces more readily transformable cells which are not well characterized in their growth regulating properties. Strongly communicating, primary epithelial cells are more characteristic of normal in- vivo conditions surrounding initiated cells. Therefore, experiments using differentiated primary cells and initiated or preneoplastic cells from the same tissue will provide the most useful information about growth and
communication in in-vivo simulating conditions. In the absence of test chemicals, there should be sufficient heterologous communication present to suppress focal growth by initiated or preneoplastic cells.
3. Cellular metabolism and culture conditions. In evaluating a test
compound as, for example, a liver promoter, it will be necessary to use cells with the metabolic and cell cycling characteristics of the organ of interest, ie, preferably primary hepatocytes. Culture materials and conditions must be chosen to prevent exposure of cells to cytotoxic, hyperplastic or growth regulating substances which alter cell cycling and differentiation properties from those observed in-vivo. Possible interactive effects of such compounds on cellular responses to GJIC regulating substances have not been studied. 4. Endpoints. For each heterologous cell system used for measurements of
GJIC, it is necessary to include direct measurements of both homologous and heterologous GJIC (via dye or radio-label transfer or metabolic cooperation)
and correlated measurements, under the same culture conditions, of an
endpoint related to proliferation of the initiated cells (such as focal growth in co-cultures or growth of initiated cells on a normal "cell mat"). A complete characterization of altered cell growth in the presence of varying proportions of normal cells (such as the type of experiment proposed in section IB)
permits a choice of heterologous culture conditions for maximum
responsiveness to GJIC effectors. All measurements of GJIC should be made after cells reach confluence, when junctional communication is maximally established (Boreiko et aL 1986). Cell growth experiments will require longer culture periods of two or more weeks for development of effects. Tests
should also be made of reversibility of observed effects upon removal of test chemical from the culture medium, with time dependence information where possible to allow for temporal correlations.
5. Dose / Response. For any chosen endpoints in a given heterologous system, and for a given test compound, as complete dose/response data as possible should be collected, from concentrations about one order of
magnitude lower than any known threshold of in-vivo promoting activity up to a minimal cytotoxic dose. All data points should result from at least two culture plates per exposure condition and high enough cell or focus counts (statistical power) to ascertain a "no effect" result.
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5. Test Substances. Each test compound should be dissolved in as small a volume as possible of DMSO, ethanol or other non-cytotoxic, water miscible solvent to be added to culture medium at no more than 0.5% by volume. In some cases, such as the exposure of lung epithelial cells to tobacco smoke, it may be desirable to provide the test compotmd or mixture in particulate form. At the end of the exposure period, the cell layer should be washed with fresh medium to remove as much residual compound as possible. All
compounds must also be tested for activity in one or more genotoxidty assays to distinguish promoting effects from growth regulatory changes caused by secondary genetic lesions.
6. Controls. Positive: Indude at least an abbreviated dose-response for a known promoter, such as TPA, in both communication and initiated cell growth assays.
Negative: Solvent controls should be included for the same exposure times as test compounds. Cytotoxidty/Plating Efficiency: The survival and colony formation rate in sparse plating trials of each cell type should be used to modifiy mixing ratios for heterologous experiments and determine upper limits for test chemical exposure.
Extracellular communication: In assays using initiated cell growth as the measured endpoint, interchange of conditioned medium between culture plates with very different proportions of initiated and normal cells will aid in detection of any focal growth regulation due to transfer of signals other than
through gap junctions.
Genotoxicity: Putative tumor promoting compounds to be evaluated for effects on GJIC and growth of initiated cells must have been shown to be non- genotoxic by one or more short term tests.
B. Development of Proposed In-Vitro Assay For Gap Jimctional Function Using the Endpoints of Focal Growth of Initiated Cells and Dye Transfer
Initial Design Rationale for Heterologous (Normal/Initiated) Communication Experiments
The work of Mehta, Bertram etal (1986), Yamasaki (1987,1988) in-vitro and
the in-vivo studies of Terzaghi-Howe (1987) and Dotto, Weinberg and Ariza (1988) have shown that normal, communicating cells can suppress the growth of preneoplastic fod (piled-up, morphologically altered cells,
descended from cells with an initial genetic lesion). Further, we know that
there is a threshold communication level below which there is no
suppression of focal growth (Mehta et al, 1986), and, if this suppression is a function only of GJIC, there will be a maximum growth suppression level corresponding to maximal heterologous communication.
Bertram (1990) plated a mixture of normal and initiated C3H/10T1/2 cells in the same proportion but at different total plating densities. The resulting higher number of foci which formed per plated initiated cell was attributed to
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the lower heterologous communication possible when cells are plated at a subconfluent density and must proliferate extensively before coming in contact with each other. A possible confounding factor in the interpretation of these resvdts is that the number of divisions required to reach confluence is higher for cells plated at lower density. Assuming a constant rate of
spontaneous mutations per generation, this would increase the probability of a second mutation capable of causing irreversible loss of growth control. If, instead, one plated different proportions of normal and initiated cells, but at a constant, nearly confluent density, in order to maximize the immediate contact of the two cell types, the following general form of communication dose/focal growth response would be expected.
fS •2 Si «j 'a, ^.^ (A
Maximum effect level
Communication