Potential role of treatment artifact in the effect of cell density upon frequencies of C3H/10T1/2 cell transformation.

Reports of unusual increases in transformation frequency in low density cultures of C3H/10T1/2 cells suggest that transformation occurs via an indirect multistage mechanism. The effect of surviving cell density upon subsequent focus production was examined in C3H/10T1/2 cultures treated with acetone, 12-O-tetradecanoylphorbol-13-acetate (TPA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNNG plus TPA, or 3-methylcholanthrene (MCA). Foci developed independent of cell density in 0.5 and 5.9% of all cultures exposed to acetone and TPA (0.25 micrograms/ml), respectively. Transformation after treatment with MNNG (0.5 micrograms/ml) occurred with low frequency (less than or equal to 7 X 10(-4)/surviving cell) and was enhanced by TPA. In MNNG plus TPA treated cultures containing less than or equal to 140 viable cells the fraction of dishes with foci was dependent upon the number of cells present at the time of MNNG treatment. As a result, relatively constant frequencies of focus formation were obtained (less than or equal to 6 X 10(-3) after correction for focus formation in TPA treated solvent controls). Focus frequency declined at cell densities greater than or equal to 350 cells. In contrast, treatment with MCA (1.0 microgram/ml) produced transformed foci with frequencies that varied from 3.3 X 10(-2) at the lowest density (5.5 cells) to 5.4 X 10(-4) at the highest (4400 cells). In low density cultures (5.6-56 cells), the fraction of dishes with foci was independent of the number of cells treated. Thus cell density had differential effects upon the frequency of foci produced by MCA or MNNG plus TPA. However, binding studies demonstrated that 6-7% of the MCA added to cell culture dishes was retained after the termination of carcinogen treatment. This residual MCA possessed biological activity which may be sufficient to elevate transformation frequencies in low density cultures.

Weak promotion of C3H/10T1/2 cell transformation by repeated treatments with formaldehyde.

C3H/10T 1/2 cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine and then repeatedly exposed to formaldehyde (0.1 to 2.0 micrograms/ml). Exposure of N-methyl-N'-nitro-N-nitrosoguanidine-initiated cultures to formaldehyde concentrations of 0.5 or 1.0 micrograms/ml in a variety of treatment regimens resulted in focus formation in up to 9% of the treated dishes. Transformed foci were observed in 2% or less of the cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine or formaldehyde alone. Formaldehyde thus appears to be only a weak tumor promoter for C3H/10T 1/2 cell transformation.

Initiation of C3H/10T1/2 cell transformation by formaldehyde.

The effects of formaldehyde were evaluated in the C3H/10T1/2 Cl 8 cell transformation system. Treatment of the cells with 0.1-2.5 micrograms/ml for formaldehyde alone did not result in significant rates of transformation. If formaldehyde treatment was followed by continuous treatment with 0.1 microgram/ml of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), transformed foci were produced. Methanol and formic acid lacked significant transforming activity under either treatment regimen. The results suggest that formaldehyde is an initiating agent for C3H/10T1/2 Cl 8 transformation. The fact that some compounds may act solely as initiators should be considered when this transformation system is used to study chemicals which may interact with cells by mechanisms similar to that of formaldehyde.

Cytogenetic analysis of spontaneous and 2-cyanoethylene oxide-induced tk-/- mutants in TK6 human lymphoblastoid cultures.

Two phenotypic classes of selectable tk-/- mutants have been isolated from the TK6 human lymphoblast cell line; one has a normal growth rate (tkn) relative to the parental cell line; the other has a slow growth rate (tks). Complete karyotypes of metaphase chromosomes were prepared and analyzed from 16 tks mutants (eight spontaneous and eight 2-cyanoethylene oxide [CNEtO]-induced), two spontaneous tkn mutants, and the parental TK6 cell line. Southern blot analysis of these tk-/- mutants indicated that all had lost a 14.8 kb polymorphic band corresponding to the active tk allele. No chromosome abnormalities with respect to the parental cell line TK6 were observed in eight spontaneous tks mutants. Chromosome abnormalities that may have been related to CNEtO treatment were observed in four of eight CNEtO mutants. However, chromosome 17, containing the tk locus in man, was cytologically normal with respect to the parental TK6 cell line in 15 of 16 tks mutants. A visible abnormality of chromosome 17 was present in one CNEtO-induced tks mutant. The abnormality was a duplication of the long arm of the chromosome 17, with break points at q11 and q21. The latter break point is close to the site of the tk locus, suggesting that the aberration observed may be associated with tk-/- phenotype. These observations contrast with the relatively high incidence (greater than or equal to 59%) of chromosome 11 abnormalities reported in tks (small colony) mouse lymphoma L5178Y/TK +/- mutants.

The use of tracheal implants in toxicology and carcinogenesis research.

Tracheal implants have served as an important experimental pathology tool with which to study the toxic and/or carcinogenic effects of chemicals upon upper respiratory tract epithelium. Initial studies with this method utilized heterotopic rat tracheal transplants which were exposed to compounds of interest, and assessed for toxic and/or carcinogenic endpoints. Grafts containing rodent tissue have proved useful for studying the cellular and biochemical features of neoplastic progression at different time intervals following in vivo exposure to carcinogens. More recent studies have utilized epithelial denuded tracheal implants inoculated with respiratory cell populations, and xenografted into immunodeficient nu/nu mice. This technique permits the study of airway epithelium from a variety of species, including man. The advent of molecular pathology techniques such as in situ hybridization will further expand the uses of tracheal implant technology for studies with xenografted human tissues. Such implants should prove useful for the examination of species- and tissue-specific characteristics of growth and differentiation by providing a bridge between cell culture and whole animal studies.

Differential effects of tumour promoters on the growth of normal human bronchial epithelial cells and human lung tumour cell lines.

The effects of the tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the colony-forming efficiency and growth of normal human bronchial epithelial (NHBE) cells and five lung squamous carcinoma cell lines were compared in medium containing 1% foetal bovine serum. TPA (0.1-5.0 ng/ml) inhibited the growth of NHBE cells and one carcinoma cell line, while four of the five carcinoma lines were less sensitive to the growth inhibitory properties of TPA but were slightly inhibited at higher TPA concentrations. The responses of NHBE cells and carcinoma cells to TPA, and the related compounds, mezerein, 4-O-methyl TPA, and phorbol were then compared in serum-free medium. In general, the removal of serum from the medium increased the differences in the responses to TPA between normal and tumour cells. Two carcinoma lines inhibited by TPA in 1% serum were stimulated by TPA in the absence of serum. Mezerein and, to a lesser extent, 4-O-methyl TPA also produced differential responses in colony-forming efficiencies between tumour lines and NHBE cells. Phorbol had no effect on either NHBE cells or on carcinoma cell lines. The relative insensitivity of carcinoma cell lines to the growth inhibitory effects of tumour promoters is consistent with the hypothesis that tumour promotion involves selection against normal cells to permit clonal expansion of preneoplastic or neoplastic cell types.

Lack of di-(2-ethylhexyl) phthalate activity in the C3H 10T 1 2 cell transformation system.

The widely used plasticizer and rodent carcinogen di-(2-ethylhexyl) phthalate (DEHP) was examined for activity in the C3H 10T 1 2 murine fibroblast cell transformation system. Treatment with DEHP or its metabolite, mono-(2-ethylhexyl) phthalate, did not produce oncogenic transformation, initiate the process of transformation in cultures treated with a tumour promoter or promote the process of transformation in cultures pretreated with a chemical carcinogen. These findings are consistent with the suggestion that the carcinogenicity of DEHP is mediated by an indirect mechanism and not by covalent interaction of DEHP with DNA.

Mechanistic aspects of initiation and promotion in C3H/10T1/2 cells.

The transformation of C3H/10T1/2 cells can be made to proceed through discrete stages of initiation and promotion. Studies of the effect of cell density upon focus formation in cultures treated with MNNG and TPA suggest that initiation by MNNG is due to a relatively infrequent, irreversible event induced by a single carcinogen treatment. In contrast, promotion appears to be a reversible process requiring multiple treatments with TPA over a protracted period of time. Some evidence suggests that promotion may entail the induction of phenotypic changes which impart a growth advantage to phenotypically unstable "initiated" cell populations. The actual cellular mechanism(s) for most of the phenomena observed in C3H/10T1/2 cultures have eluded precise definition and widely divergent hypotheses have been advanced to explain transformation, initiation, and promotion. Conceivably there are multiple mechanisms responsible for each of these phenomenon. Some agents may transform by a multistage mechanism whereas others may exert their effects in a more direct fashion. Some of the foci produced by promotion may be the result of simple selective processes, others the product of more complex inductive events. Variations would thus be expected between laboratories working with different protocols and agents. As demonstrated by the possible involvement of an MCA residue in transformation, it is also apparent that fundamental technical aspects of this conceptually simple cell transformation system are poorly understood. While it is natural to develop mechanistic models based on quantitative observations of transformation, a limited understanding of the basic cell culture variables which modulate both the induction and expression of transformation dictate that caution be exercised in extrapolating the significance of such models to in vivo carcinogenesis.

Initiation and promotion in cultures of C3H10T1/2 mouse embryo fibroblasts.

Studies conducted in numerous laboratories have demonstrated that the transformation of C3H10T1/2 cells can proceed through discrete stages of initiation and promotion. Indeed, multiple operational aspects of initiation and promotion in this system closely mimic the essential characteristics of initiation and promotion on mouse skin. The sensitivity of this system to the effects of different tumor promoters also appears to parallel that of mouse skin, and there is evidence to suggest that the C3H10T1/2 system is most sensitive to agents acting as stage II tumor promoters on mouse skin. Sensitivity to compounds active at other tissue sites in rodents and perhaps man has also been observed. At this time it is difficult to assess the relevance of the C3H10T1/2 system for the study of agents capable of modulating respiratory carcinogenesis. The process of promotion can possess extreme tissue and species specificity and effects observed in murine fibroblasts of embryonic origin may have little practical bearing upon effects to be anticipated in the tracheal epithelium of the rat or the bronchial epithelium of man. This is not to say that the C3H10T1/2 system is irrelevant to respiratory carcinogenesis. However, due recognition must be taken of the probable natural limitations of this system for the study of promoters of respiratory carcinogenesis. As the data base for the use of this system is expanded, the relationship between promotion in C3H10T1/2 cells and the respiratory tract of man and rodents will become better defined. Until such time as this relationship is firmly established, it is perhaps best to regard the C3H10T1/2 system as an interesting model with which results obtained using respiratory tissue can be compared or contrasted.

Promotion of C3H/10T1/2 morphological transformation by polychlorinated dibenzo-p-dioxin isomers.

Few of the seventy-five chlorinated dibenzo-p-dioxin isomers present in the environment have been adequately characterized for their carcinogenic potential. In previous studies we observed that the carcinogenic dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin promoted cell transformation when continuously applied to C3H/10T1/2 cells initiated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The current study was undertaken to evaluate the response of the C3H/10T1/2 cell transformation system to several other dioxin isomers of known carcinogenic potential. 1,2,3,6,7,8- and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (HCDD) (1-1000 nM) and 2,7-dichlorodibenzo-p-dioxin (0.1-20 microM) failed to transform C3H/10T1/2 cells or to initiate transformation in cultures subsequently treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Continuous exposure of MNNG-initiated cultures to 2,7-DCDD (1-5000 nM) produced elevated but not statistically significant numbers of transformed foci at the highest dose tested. 1,2,3,6,7,8- and 1,2,3,7,8,9-HCDD were promoters of transformation when applied at concentrations greater than or equal to 12 and 40 pM, respectively, to C3H/10T1/2 cultures initiated with MNNG. Maximum responses for both HCDD isomers were attained at concentrations between 120 and 400 pM. These studies suggest that the C3H/10T1/2 cell transformation system may provide a relevant in vitro model for the identification and study of carcinogenic dioxin isomers.

Limiting factors of the V79 cell metabolic cooperation assay for tumor promoters.

A number of chemicals which promote tumorigenesis in vivo have been observed to inhibit metabolic cooperation between 6-thioguanine-resistant (TGR) and sensitive (TGS) Chinese hamster lung V79 cells. The apparent correlation between an inhibition of metabolic cooperation in V79 cells in vitro and promotion of oncogenesis in vivo has led to the suggested utilization of the assay as a screen for tumor promoters. However, many features of the V79 metabolic cooperation assay which would provide for an improved understanding of the usefulness and limitations of the assay have not been well characterized. A number of experimental parameters involved in the metabolic cooperation assay were examined with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA inhibited metabolic cooperation between V79 cells in a dose-dependent manner, and thereby increased the recovery of TGR cells co-cultured with TGS cells in the presence of 6-thioguanine. Approximately 90% recovery of TGR cells was achieved at TPA concentrations of 1--1000 ng/ml, a 9- to 11-fold higher yield than the average background recovery of 8--10% in acetone-treated cultures. The TPA-induced inhibition of metabolic cooperation was observed to be transient. Pretreatment of TGS and TGR cells with TPA for 12 h or more prior to 6-thioguanine addition resulted in no inhibition of metabolic cooperation. It was further determined that the presence of TPA was required for only 1 h to maximally inhibit metabolic cooperation, a significantly reduced period of exposure relative to the originally proposed 4 day exposure. Technical grade dinitrotoluene (DNT), 2,4-DNT and 2,6-DNT which have demonstrated promoting activity in rat liver did not increase the recovery of TGR cells. However, the solvent dimethylsulfoxide (DMSO) at concentrations ranging from 1.0 to 2.5% increased the recovery of TGR cells in a dose-dependent manner. The short-lived effect of TPA suggests that inhibition of metabolic cooperation may not bear a mechanistic relationship to tumor promotion. The inhibition of metabolic cooperation by DMSO, the requirement for only short-lived reductions in metabolic cooperation for maximal TGR cell recovery, and the lack of inhibition by DNT suggests that caution should be exercised when interpreting the results of this bioassay.

Alterations of intercellular communication associated with the transformation of C3H/10T1/2 cells.

Cultures of C3H/10T1/2 mouse embryo cells were treated in accordance with several treatment regimens that induced the focal growth of morphologically transformed cells. Intercellular communication between focus cells, and between focus and monolayer cells, was examined in late stages of transformation experiments by microinjection of Lucifer yellow dye into cells and observation of dye transfer to surrounding cells. Transformed foci produced by treatment with 3-methylcholanthrene, by initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), or by initiation with MNNG and promotion with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. These included focus types thought to possess tumorigenic potential (Types II and III) and those believed to lack such potential (Type I). Cells within all focus types exhibited only limited communication with each other or with surrounding monolayer cells. In contrast, microinjection of monolayer cells typically resulted in dye transfer to an average of approximately 50 other monolayer cells. The presence of the tumor promoters TPA or TCDD did not alter intercellular communication between monolayer cells. These studies demonstrate that alterations in intercellular communication are evident during the growth of transformed foci. These changes are relatively independent of both the treatment regimen used to produce foci and the presumed oncogenic potential of different focus types.

Reversible expression of morphological transformation in C3H/10T1/2 mouse embryo cultures exposed to 12-O-tetradecanoylphorbol-13-acetate.

Treatment of C3H/10T1/2 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine and then 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the production of numerous foci of morphologically transformed cells. When dishes containing foci were provided medium which did not contain TPA, up to 84% of the foci were found to regress. Promotion of morphological transformation by TPA in C3H/10T1/2 cells may thus be a reversible process.

Analysis of prospective epidemiologic studies on the neurobehavioural effects of lead.

High-level lead exposure can have serious effects on the intellectual and behavioural development of young children. There has been much controversy in the last decade concerning the possible impact of low-level lead exposure upon the neurobehavioural and psychomotor development of children. Five longitudinal studies (Boston, Cincinnati, Cleveland, Port Pirie and Sydney) examining lead effects on child development were initiated in the early 1980s. These studies share multiple design features and include data on blood lead and neurobehavioural measurements from birth, six months, or annual intervals to seven years. All the studies use multivariate analysis to take into account possible confounding covariates with outcome measures.The studies tend to have varying results based on the covariates used and type of subject population. An analysis of the results of the five studies with regard to effects associated with prenatal and postnatal lead exposure and pregnancy outcome has been carried out and reveals inconsistencies in the onset, stability, and nature of neurobehavioural effects correlated with different indices of lead exposure. it is possible that the variation in reported results may be due to the use of different covariates and analyses among studies. A common analysis should be carried out among ail studies to further determine consistency of results. Although individual studies may show some effects, taken as a whole, the current published data from the five studies in this review are inconsistent and do not lend support to the concept that low level lead exposure resulting in blood lead levels below 25 µg dL(-1) is associated with neurobehavioural deficits in children.

Factors influencing the promotion of transformation in chemically-initiated C3H/10T1/2 Cl 8 mouse embryo fibroblasts.

Treatment of low density asynchronous cultures of C3H/10T1/2 Cl 8 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiates the process of transformation and produces significant numbers of transformed foci only when treated cultures are subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell culture variables which influence the outcome of this initiation and promotion system were studied. A TPA concentration of 0.25 micrograms/ml was found to be optimal for the promotion of focus production and the presence of TPA was required both during logarithmic growth and throughout confluence. The lot of fetal calf serum used to cultivate the cells also played a determining role in focus production. Of nine serum lots purchased from four different suppliers, only two were suited for initiation and promotion studies with MNNG and TPA. In contrast, seven of these lots were adequate for transformation studies with 3-methylcholanthrene. Factors which adversely influenced focus production included the use of fungizone or the use of high passage stock cultures. These studies demonstrate that cell culture variables which influence promotion in these cells can be controlled and that this system can be successfully used in studies of the cellular mechanism of in vitro promotion and for the detection of genotoxic substances.

Role of intercellular communication in the promotion of C3H/10T1/2 cell transformation.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) upon intercellular communication and promotion were studied in cultures of C3H/10T1/2 mouse embryo fibroblasts. Cell-to-cell communication was quantitated by autoradiographic analysis of [3H]uridine transfer from prelabelled donor cells to an excess of unlabelled recipient cells during a 4 h co-cultivation. Extensive transfer of label was observed from donor to recipient cells in contact. Treatment of non-transformed C3H/10T1/2 cultures with 250 ng/ml TPA at co-cultivation of donor and recipient cells markedly inhibited intercellular communication during the 4 h incubation, producing an 80% reduction in uridine exchange relative to solvent-treated control cultures. Concentrations of TPA ranging from 0.25 to 25 ng/ml were also effective in inhibiting [3H]uridine exchange in a dose dependent fashion from 13% to 74%. This inhibition of intercellular communication was transient; cells exposed to 250 ng/ml TPA for 1.5 h prior to co-cultivation with TPA exhibited a 60% inhibition and the exchange of uridine had increased to control values in cultures pretreated for 12-72 h. An examination of label transfer between non-transformed and transformed C3H/10T1/2 cells indicated that both the extent of inhibition by TPA and the kinetics of communication inhibition were similar to that observed for non-transformed cells. Initiation and promotion experiments demonstrated that exposure to 250 ng/ml TPA for 5 weeks, but not to reduced concentrations of 0.25, 2.5 or 25 ng/ml, was capable of promoting morphological transformation. The lack of correlation between the dose responses of TPA for promotion and for reduction of cell-to-cell communication, and the transient nature of intercellular communication inhibition by TPA, suggests that an inhibition of cell-to-cell communication is not a sufficient event for promotion of oncogenic transformation in these cells.

Enhanced sensitivity of the C3H/10T1/2 cell transformation system to alkylating and chemotherapeutic agents by treatment with 12-O-tetradecanoylphorbol-13-acetate.

The failure of the C3H/10T1/2 cell transformation system to respond to numerous known carcinogens has limited its applications for the detection and study of cancer-causing substances. Recent studies have found, however, that some carcinogens function as initiating agents for the process of transformation in these cells. Treatment with such agents is generally not sufficient to transform low-density asynchronous cultures of C3H/10T1/2 cells, but morphologic transformation will occur if such cultures are subsequently exposed to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, the ability of TPA to enhance transformation was examined in cultures treated with a variety of chemical agents. The addition of TPA after chemical treatment enhanced the transformation of these cells by methylmethanesulfonate, ethylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, N-nitrosomethylurea, N-nitrosoethylurea, mitomycin C, 5-fluorodeoxyuridine, and 5-azacytidine. Treatment with amethopterin or benzo(e)pyrene did not produce significant numbers of foci in the presence or absence of TPA. TPA inhibited transformation by high concentrations of 3-methylcholanthrene and benzo(a)pyrene. Thus, numerous carcinogens function as initiating agents for these cells and the presence of TPA can dramatically increase the sensitivity of this cell transformation system.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2 cells.

Continuous treatment of C3H/10T1/2 cells with low concentrations (greater than or equal to 4 pM) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhanced focus production in cultures pretreated with N-methyl-N'-nitro-N-nitrosoguanidine. Maximal enhancement occurred at 40 pM TCDD, a concentration 10 000-fold lower than that required to produce an optimal response with 12-O-tetradecanoylphorbol-13-acetate. Single treatments with 0.06 nM-5 microM TCDD did not transform C3H/10T1/2 cells or initiate the process of transformation in cultures subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Promotion of transformation is thus the predominant effect of TCDD in the C3H/101/2 cell transformation system.

Cytotoxic and cytogenetic effects of asbestos on human bronchial epithelial cells in culture.

Asbestos and other mineral fibers elicit responses in several rodent cell transformation systems. The mechanism of this transformation has been hypothesized to involve specific chromosome alterations, especially changes in chromosome number. However, the cytogenetic effects of asbestos fibers in cultured human respiratory epithelium have not been well characterized. The present study examined the effects of chrysotile and crocidolite asbestos fibers on cultures of human bronchial epithelial (HBE) cells growing in serum-free medium. HBE cells were continuously treated with chrysotile (0-4 micrograms/cm2) or crocidolite (0-300 micrograms/cm2) asbestos and examined after 24, 48, 72 or 96 h for cytotoxic and cytogenetic effects. Both asbestos fiber types induced a concentration-dependent inhibition of cell proliferation and colony-forming efficiency; however, in these assays chrysotile was 100-300 times more toxic than crocidolite. Concentrations of asbestos that inhibited growth had little effect upon trypan blue exclusion or intracellular esterase activity, suggesting that the majority of asbestos-exposed cells were still viable. A 2.7-fold increase in binuclei and a 1.6-fold increase in micronuclei were observed 72 h after treatment with 4 micrograms/cm2 chrysotile. A 1.9-fold increase in binuclei was observed 72 h after treatment with 300 micrograms/cm2 crocidolite, but crocidolite did not increase the incidence of micronuclei. Chrysotile asbestos failed to induce significant numerical chromosome changes in HBE cells and increased structural aberrations only at the 24 h time point. These findings contrast with the relatively high incidences of asbestos-induced chromosome changes previously observed in some rodent cell cultures and suggest the existence of species-specific or cell-type-specific differences in either chromosome stability or mechanism(s) of asbestos-induced toxicity.

Effect of mouse skin tumor promoters upon [3H]uridine exchange and focus formation in cultures of C3H/10T1/2 mouse fibroblasts.

The abilities of 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA) and mezerein to promote the process of transformation were evaluated in cultures of C3H/10T1/2 mouse embryo fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine. Mezerein was found to be as potent as the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) for the promotion of focus formation, eliciting a promotion response at concentrations that ranged from 100 to 2500 ng/ml. 4-O-Methyl-TPA (25-2500 ng/ml) did not promote focus formation, but was mitogenic for confluent cultures. The effects of promoting and non-promoting compounds upon intercellular communication were then evaluated to determine if a rapid assay for the inhibition of communication might serve as a surrogate for the relatively long term, labor-intensive cell transformation assay. Inhibited intercellular communication, as measured by inhibition of [3H]uridine exchange between cells, appeared to correlate with the ability of phorbol related compounds to promote transformation. However, the potent promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit [3H]uridine transfer. Inhibition of intercellular communication may thus be diagnostic of the promoting potential of phorbol-related compounds in C3H/10T1/2 cultures, but may not be an appropriate endpoint for the study of carcinogenic dioxins.