Evaluation of the V79 cell metabolic co-operation assay as a screen in vitro for developmental toxicants.

Inhibition of intercellular communication is proposed to be one of several possible mechanisms of teratogenesis. 38 coded compounds were tested for their effect on intercellular communication in the V79 cell metabolic co-operation assay. Test chemicals were selected from a list of 47 agents recommended for the evaluation of assays in vitro for developmental toxicants. In addition to testing the effects of chemicals on intercellular communication, a separate cytotoxicity assay determined the concentration of each chemical that inhibited clonal expansion of V79 cells. Seven of the 29 designated teratogens were positive for inhibition of intercellular communication in the V79 assay. Additionally, four teratogens and one non-teratogen inhibited intercellular communication at only a single concentration or at cytotoxic concentrations and were scored as equivocal. Therefore, the sensitivity of the V79 assay for teratogens was 24% (seven of 29 teratogens tested positive), or 38% if the four equivocal chemicals are considered positive. None of the nine non-teratogens unequivocally inhibited intercellular communication, resulting in a specificity of 100%, which decreased to 89% when the single equivocal score was considered positive. The overall accuracy for correctly identifying teratogens and non-teratogens was 42% when equivocal chemicals were considered negative, and 50% if they were considered positive in the V79 assay. The results demonstrate that despite relatively low accuracy regarding a diverse group of developmental toxicants, chemicals that did inhibit intercellular communication under the present conditions had a high probability of being a teratogen. The low accuracy reported here contrasts with earlier reports on the assay and possible reasons for this are discussed.

Inhibition of intercellular communication in Chinese hamster V79 cells by fractionated asphalt fume condensates.

Asphalt fume condensate is a skin carcinogen in mice, yet this complex mixture contains relatively low levels of known carcinogenic initiators. Consequently, its biological activity has been attributed to the presence of cocarcinogenic or tumor-promoting agents. One of several proposed mechanisms of tumor promotion is inhibition of intercellular communication. In an attempt to determine if asphalt fume has tumor-promoting potential inhibition of intercellular communication was measured in V79 cells exposed to fractionated asphalt fume condensate. Fume from air-blown Arabian crude asphalt was trapped and separated into five fractions by preparative-scale high-pressure liquid chromatography. The parent fume condensate and the five fractions inhibited intercellular communication in a concentration-dependent fashion, with a minimum effective concentration of 2.5 microgram/ml for the most potent fraction. Cytotoxicity assays were performed at the same time and concentrations as the metabolic cooperation assays. Cytotoxic responses paralleled the inhibition of intercellular communication.

Interlaboratory studies with the Chinese hamster V79 cell metabolic cooperation assay to detect tumor-promoting agents.

Three laboratories participated in an interlaboratory study to evaluate the usefulness of the Chinese hamster V79 cell metabolic cooperation assay to predict the tumor-promoting activity of selected chemicals. Twenty-three chemicals of different chemical structures (phorbol esters, barbiturates, phenols, artificial sweeteners, alkanes, and peroxides) were chosen for testing based on in vivo promotion activities, as reported in the literature. Assay protocols and materials were standardized, and the chemicals were coded to facilitate unbiased evaluation. A chemical was tested only once in each laboratory, with one of the three laboratories testing only 15 out of 23 chemicals. Dunnett's test was used for statistical analysis, and differences between treated- and control-cell responses were analyzed at P less than or equal to .01. Chemicals were scored as positive (at least two concentration levels statistically different than control), equivocal (only one concentration statistically different), or negative. For 15 chemicals tested in all three laboratories, there was complete agreement among the laboratories for nine chemicals. For the 23 chemicals tested in only two laboratories, there was agreement on 16 chemicals. With the exception of the peroxides and alkanes, the metabolic cooperation data were in general agreement with in vivo data. However, an overall evaluation of the V79 cell system for predicting in vivo promotion activity was difficult because of the organ specificity of certain chemicals and/or the limited number of adequately tested nonpromoting chemicals.

Neurogram recording during acute inflammation in the rat hind paw.

Electrophysiological recording was conducted using the intact tibiotarsal nerve of the left hind leg of adult male rats before and after subplantar injections of three different phlogistic agents or saline vehicle in the left hind paw. In three-hour recordings, peak neural activity as activated by 5 g of pressure to the injected paw was reached in 90 min (dextran), 120 min (brewer's yeast) and 150 min (carrageenan). The least neural hyperactivity was seen with dextran, while yeast and carrageenan produced comparably high degrees of hyperactivity. Neurograms were also studied in rats receiving orally administered prototype anti-inflammatory agents or chlorpromazine 1 hr before carrageenan was injected pedally. Chlorpromazine HCl pretreatment (100 mg/kg) exerted the greatest protective effect with the nadir of neural activity seen at 90-120 min. The nadir for phenylbutazone (100 mg/kg) was at 60-120 min, indomethacin (10 mg/kg) at 60-90 min and aspirin (300 mg/kg) at 45-90 min. Despite these temporal differences, the protective effects of these three anti-inflammatory agents were statistically equivalent at the doses tested. Hydrocortisone alcohol (20 mg/kg) provided a significant reduction in hyperactivity at 30-60 min but the duration was much shorter than the other agents which showed protective effects through to the end of the 180-min observation period.

Improved conditions for murine epidermal cell culture.

An improved method for cultivating newborn mouse epidermal cells has been developed that increases the longevity, epithelial nature and efficiency of cell-line establishment. The use of Super Medium, an enriched Waymouth's formulation, increased proliferation for long periods of time, as did incubation at 31 degrees C rather than 37 degrees C. The fetal bovine serum requirement was found to be reduced at the lower temperature. An increase in labeling indices was seen when epidermal growth factor (EGF) or the cyclic nucleotides were added and the presence of EGF receptors was determined. Of the prostaglandins (PG) examined, PGE1 and PGE2 produced the greatest increase in DNA synthesis. The PG precursors, arachidonic and 8,11,14-eicosatrienoic acid, were also greatly stimulatory. The use of a lethally irradiated 3T3 feeder layer at 31 degrees C proved superior in maintenance of an epithelial morphology. Subculturable cell lines were established much more readily and reproducibly in carcinogen-treated cultures grown under the improved conditions.

Prostaglandin modulation of phorbol ester skin tumor promotion.

TPA promotion of skin tumors in mice can be modified by application of various prostaglandins or their precursors. The effects depend on the particular prostaglandin used: PGF2alpha enhances promotion, whereas PGE1 consistently inhibits promotion. Time of application of the prostaglandin with respect to TPA determines whether PGE2 enhances or inhibits. Dose-dependent inhibition was observed for arachidonic acid. The prostaglandins alone were unable to elicit tumors in initiated mice.