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Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters., (Copyright 2001 Academic Press.)

Photodynamic treatment (PDT) of confluent MDCK II cells resulted in a noticeable clustering of dead cells, consistent with a significant bystander effect. Likewise, PDT of cells in microcolonies resulted in an overabundance of microcolonies that had responded to the treatment as a single unit, that is, in which either all or no cells were dead. Confluent MDCK II cells appeared to communicate via gap junction channels, while cells in microcolonies did not. Monte Carlo simulation models were fitted to the distributions of dead cells in confluent monolayers and in microcolonies. The simulations showed that the degree of the bystander effect was higher in microcolonies than in confluent cells, suggesting that gap junction communication may be involved in the bystander effect. However, when the gap junction hypothesis was tested by treatment of microcolonies with 30 microM dieldrin, an inhibitor of gap junctional intercellular communication, there was no reduction of the bystander effect, indicating that this effect was not mediated by gap junctional intercellular communication. PDT influenced phosphorylation of tyrosine residues in several proteins in the cells. Protein phosphorylation is important in cellular signaling pathways and may be involved in the bystander effect, for example by influencing the mode of cell death.

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells., (Copyright 1999 Academic Press.)

A monoclonal antibody, Zymed 13-8300, was previously reported to only detect nonphosphorylated connexin43 (Nagy et al., Exp. Cell Res. 236, 127-136, 1997). We show that 13-8300 can detect several phosphorylated species of connexin43 in Western blots after stimulation of two fibroblast cell systems with fresh growth medium, 12-O-tetradecanoyl phorbol-13-acetate, pervanadate, or permolybdate. In one of the cell systems, at least three forms of phosphorylated connexin43 could migrate at the same position during electrophoresis. The comigration of differentially phosphorylated species may complicate the molecular and functional analysis of phosphorylation sites in Cx43. Immunofluorescence experiments indicated that the newly generated phosphorylated Cx43 forms mainly had a perinuclear location. Also, in cells treated with brefeldin A for 8 h, in which the majority of connexin43 was intracellular, phosphorylation was induced by the agents. Phosphorylation of intracellular connexin43 can therefore be induced by several stimuli., (Copyright 1999 Academic Press.)

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5-15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding.

Biological and analytical characterizations of permolybdate (a mixture of H2O2 and molybdate) were done. Molybdate (10 mM) and molybdenum(V) chloride (3 mM) did not affect gap junctional intercellular communication (GJIC), phosphorylation status of connexin43 (Cx43) or cellular tyrosine phosphorylation in early passage hamster embryonic cells (mainly fibroblast-like). High concentrations of H2O2 (3-10 mM) affected some of the parameters. Acidified permolybdate was clearly more stable than the unadjusted permolybdate. The maximum biological potency of acidified permolybdate was found at a molar ratio of 2:1 (H2O2:molybdate). The mixtures of molybdenum(V) chloride and H2O2 gave a maximum effect at 4:1 molar ratio (H2O2:molybdenum(V)). This can be explained by decomposition of H2O2 and by the generation of less biologically active compounds. Spectrophotometric analyses of the mixtures corroborated the biological results. The Mo(V) electron spin resonance spectrum disappeared upon addition of H2O2 to Mo(V) solutions, and no spectrum appeared when H2O2 was mixed with Mo(VI). Thus, permolybdate is probably diperoxomolybdate, a Mo(VI) compound. Regardless of the parent metal salt, the H2O2/metal salt mixtures showed concentration-dependent biphasic responses with an initial decrease in GJIC followed by an increase. A dissociation between alteration in Cx43 phosphorylation status and GJIC was obtained under certain conditions. The biological activities of permolybdate were only partially mimicked by phenylarsine oxide, an alternative protein tyrosine phosphatase inhibitor.

The protein-tyrosine phosphatase inhibitors pervanadate, permolybdate, H2O2, and to a much lesser extent vanadate, increased the amount of cellular phosphotyrosine and induced tyrosine phosphorylation of connexin43 (Cx43) in early passage hamster embryo fibroblasts. The presence of phosphotyrosine in Cx43 immunoprecipitates from pervanadate-treated cells was shown by a phosphotyrosine-specific antibody and a phosphotyrosine-specific phosphatase. Pervanadate-induced Cx43 tyrosine phosphorylation was further verified by phosphoamino acid analysis, while no phosphotyrosine was present in control cells. This is the first observation of tyrosine phosphorylation of connexins in normal cells.

Gap junctional intercellular communication (GJIC), phosphorylation status of the gap junction protein, connexin43 (Cx43), and cellular tyrosine phosphorylation in Syrian hamster embryo cells have been employed for a biological characterization of pervanadate (a mixture of H2O2 and vanadate). In addition, electron paramagnetic resonance (EPR) spectroscopy was used to follow the appearance and disappearance of vanadyl (V(IV)). It has previously been suggested that pervanadate is vanadyl hydroperoxide (V(4+)OOH). This assumption was tested by using mixtures with different molar ratios of H2O2 and orthovanadate, metavanadate or vanadyl sulfate. The maximal biological activity of the mixtures were found at a molar ratio of 2:1 (H2O2:orthovanadate or metavanadate) or 2.5:1 (H2O2:alkaline vanadyl sulfate). No V(IV) EPR spectrum appeared upon mixing orthovanadate or metavanadate and H2O2. The V(IV) EPR spectrum disappeared when vanadyl sulfate was incubated with H2O2 in a 0.5:1 molar ratio (H2O2:alkaline vanadyl sulfate). Spectrophotometrically, a V(V)-like peak at 265 nm had its optimum at this ratio. These results are consistent with pervanadate being diperoxovanadate. The individual compounds were prominently less active than the per-compound mixtures in affecting the biological parameters. The decreases in GJIC showed a concentration-dependent correlation with the onset of the alterations of the Cx43 band pattern and the amount of phosphotyrosine in cellular proteins, but the correlation was not absolute. All the studied biological parameters were reversible, also under continuous exposure to pervanadate.

The effects of K2CrO4, H2O2, benzoyl peroxide, menadione, KBrO3 and UV365nm on gap junctional intercellular communication (GJIC) have been studied in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive Syrian hamster embryo (SHE) cell line BPNi. All agents were found to increase the level of GJIC by 50-100%. Also, in early passage SHE cells, a tendency for increased GJIC was found for the oxidative agents studied. Hydrogen peroxide was used as a model compound in the subsequent studies. The increase in GJIC was reversible, and it was not due to an increased non-junctional permeability. Hydrogen peroxide counteracted the TPA-induced decrease in GJIC, regardless of whether the cells were exposed to the compounds simultaneously or the cells were pre-exposed to TPA before addition of H2O2. The GJIC enhancement by H2O2 was slightly reduced by the addition of the hydroxyl radical scavenger dimethylsulphoxide or by the inhibition of catalase by amitrole. The cAMP/protein kinase A system is the only characterized signal transduction system that is known to increase GJIC in most cell types. Hydrogen peroxide did not increase the amount of cAMP (or cGMP) in BPNi cells, while forskolin and a phosphodiesterase inhibitor had to increase the cAMP level several-fold to affect GJIC to the same degree as the oxidative agents. Some inhibitors of protein kinase A were assayed for their ability to inhibit the increases in GJIC caused by H2O2 and forskolin. Staurosporine inhibited the forskolin-induced increase in GJIC, with much less effect on the H2O2-induced increase. H8, H88 and H89 had less effect than staurosporine on the forskolin-induced increase in GJIC. The results suggest that the cAMP/protein kinase A system may not be involved in the increase in GJIC caused by H2O2, although this cannot be completely ruled out.

The effects of ultraviolet (UV) radiation on gap junctional intercellular communication (GJIC) in V79 Chinese hamster fibroblasts were studied by means of a dye transfer assay. Intercellular communication was shown to be altered by UVB (297/302 nm) and UVA (365 nm) radiation, the effect depending on the wavelength of exposure and time between irradiation and microinjection of the dye in the dye transfer assay. Exposure to 297/302 nm radiation induced a reduction in intercellular communication 6 min after exposure. Incubation of the cells post-irradiation reversed the inhibition of GJIC. From 2 to 24 h after exposure an increase in GJIC over the control cells was seen, with a maximum at 8 h post-irradiation. UVA (365 nm) radiation, on the other hand, induced an increase in the intercellular communication 6 min after irradiation. Incubation of the cells post-irradiation led to a decrease in the number of communicating cells, with a minimum seen 4 h after exposure. The reduction in communication observed after exposure to UVB and UVA was not correlated with similar modifications in the gap junction protein connexin43 as found when exposing the cells to the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate. For the higher fluences of UVA, a decrease in immunorecognizable connexin43 was seen, concomitant with a markedly increased background of higher mol. wt compounds. This may be due to UVA-induced crosslinking of connexin43. No correlation was found between changes in communication induced by UV radiation and levels of cyclic AMP.

A number of phosphatase inhibitors (okadaic acid, calyculin A, aluminium fluoride, sodium molybdate, sodium orthovanadate, pervanadate and vanadyl sulphate) were investigated for their effects on gap junctional intercellular communication (GJIC) and [125I]-epidermal growth factor (EGF) binding in early passage Syrian hamster embryo cells (mainly fibroblast-like cells) and in V79 Chinese hamster lung fibroblasts. Only pervanadate decreased GJIC significantly. After the initial pervanadate-induced decrease the GJIC recovered rapidly. Only pervanadate was able to change the band pattern of the gap junction protein connexin43 (cx43) in Western blots. Together this may indicate either that there is a low turnover of phosphate groups in cx43 under basal conditions or that the putative phosphatases are not sensitive to most of the phosphatase inhibitors applied. In contrast, pervanadate, orthovanadate and molybdate decreased [125I]-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is able to induce the phosphorylation of both cx43 and the EGF receptor, concomitantly with a decrease in GJIC and [125I]-EGF binding. These effects are reversible after removal of TPA. It could be imagined that other phosphatases would act on cx43 and the EGF receptor after the forced phosphorylation of the two molecules. Thus TPA was used to downregulate GJIC and [125I]-EGF binding and phosphatase inhibitors were applied in the upregulation phase. Only pervanadate affected the upregulation of GJIC, and pervanadate, orthovanadate and molybdate affected the upregulation of [125I]-EGF binding. Thus it is not an identical complement of phosphatases that act on cx43 and the EGF receptor. All the downregulating agents are assumed to be phosphotyrosine phosphatase inhibitors.

A study was made of whether normal and morphologically transformed colonies in the Syrian hamster embryo (SHE) cell transformation assay performed heterologous gap junctional intercellular communication (GJIC). Two compounds, 12-O-tetradecanoylphorbol-13-acetate (TPA) and Na-orthovanadate (vanadate), which induce high frequencies of morphological transformation in SHE cells, have been employed. Three approaches were used to study the possibility of heterologous GJIC. (i) Morphologically transformed colonies (induced by TPA) partially overlapping with normal colonies were selected. Cells in the border area were micro-injected with the fluorescent dye Lucifer yellow to determine whether the dye spread to cells belonging to the other colony. This approach proved to be unsuccessful due to an inability to pinpoint which cells belonged to which colony. (ii) X-irradiated, non-dividing feeder cells are easily recognized by their large size. Feeder cells in contact with normal or TPA-transformed colonies were injected with Lucifer yellow. The dye was found to spread to most of the contacting cells, irrespective of whether they belonged to a normal or morphologically transformed colony. (iii) TPA- and vanadate-exposed colonies were labelled by endocytosis of Lucifer yellow overnight. This resulted in a punctate fluorescent pattern. Unlabelled, previously unexposed cells were seeded onto the dishes and incubated for 3.5-7 h. The ability to perform heterologous GJIC between the newly seeded cells and labelled colony cells was investigated. Both normal and transformed colonies were found to be able to communicate with the newly seeded cells. Thus, the present results indicate that selective communication is not a general property of morphologically transformed SHE cell colonies.

Coxsackie B1 virus induces increased susceptibility to invasion by Shigella flexneri when HEp-2 cells are inoculated with the complete virus. When RNA from the same virus was microinjected into cells, virus RNA was synthesized and new virus particles were formed, but the transfected RNA had no effect on bacterial invasiveness. However, when the cells were prestimulated with UV-inactivated virus, the microinjected RNA induced an additional enhancement of bacterial invasiveness. Microinjected whole virus particles did not replicate and did not induce any change in bacterial invasiveness. The results indicate that an initial event in virus multiplication is necessary to achieve an effect of transfected viral RNA on invasion of S. flexneri.

The levels of gap junctional intercellular communication (GJIC) were studied in normal, morphologically altered and morphologically transformed colonies formed in the Syrian hamster embryo (SHE) cell transformation assay. The colonies were selected from non-exposed dishes or dishes exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.16 microM), di(2-ethylhexyl)phthalate (DEHP, 77 microM), Na-orthovanadate (vanadate, 3.4 microM) or dieldrin (25 microM) for 7 days during colony formation. TPA, DEHP and vanadate induced increased frequencies of morphological transformation of colonies. At the same time, TPA and DEHP decreased GJIC in the colonies by approximately 30% under the conditions used. All categories of colonies were equally affected. Vanadate did not change the level of GJIC in any of the categories of colonies compared to unexposed control. Dieldrin strongly suppressed GJIC in all colonies without increasing the frequency of transformation. The compounds affected GJIC after short-term exposures (4 and 24 h) to cell monolayers rather similarly to that found after long-term exposure to the colonies. Transformation assays with coexposure of dieldrin together with the transforming agents vanadate, DEHP or benzo[a]pyrene did not increase transformation frequencies compared to the transforming agents alone. The GJIC level in all coexposure groups was similar to that of dieldrin alone. Furthermore, regardless of whether dieldrin was present or not, removal of vanadate 24 h before fixation of the colonies caused a slight decrease in the transformation frequency. The results suggest that: (i) morphologically transformed colonies have the same ability of intercellular communication as normal colonies; (ii) decreased GJIC is probably not either sufficient or necessary to induce transformation of SHE cell colonies; (iii) a decreased level of GJIC does not necessarily increase the susceptibility of SHE cells for transformation; and (iv) inhibition of GJIC may not have an impact on the maintenance of the transformed phenotype of SHE cell colonies.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPP A), sapintoxin D (SAP D) and sapintoxin A (SAP A) on the decrease in [125I]epidermal growth factor (EGF) binding (indicating protein kinase C activation), suppression of gap junctional intercellular communication (GJIC) and induction of morphological cell transformation (MCT) in Syrian hamster embryo (SHE) cells were investigated. All five phorbol esters were found to reduce [125I]EGF binding in early passage SHE cells at comparable concentrations. DOPP A was approximately 10-fold less potent in decreasing GJIC compared to the other phorbol esters in early passage SHE cells, while the compounds showed less difference in suppressing GJIC in the phorbol ester sensitive SHE cell line BPNi. The decreases in [125I]EGF binding and GJIC were found to be transient in the continuous presence of phorbol esters. All phorbol esters induced MCT in early passage SHE cells, but DOPP and DOPP A were approximately 10-fold less potent than TPA, SAP D and SAP A. Thus, there seems to be some degree of correlation, but not to a full extent, between the ability of the phorbol esters to activate PKC, decrease GJIC and to induce MCT. The results do not suggest a simple relationship between PKC activation, inhibition of GJIC and the reported tumor-promoting activities of the compounds.

Coxsackie B1 virus infection enhances the susceptibility of cultured HEp-2 cells to Shigella flexneri invasiveness. This can be reproduced partially with UV-inactivated virus, particularly the effect observed shortly after viral inoculation. The following phases of viral multiplication were correlated with the enhancing effect: uncoating of viral particles, synthesis of viral RNA and proteins, and assembly of newly produced virus particles. Uncoating of virus particles was completed within 60 min. This process was not correlated with the development of the early effect on invasiveness. Intact virus capsids seem to be necessary to enhance bacterial invasiveness in the early phase of virus infection. Separated capsid proteins had no effect either when applied to the cell surface or when microinjected into the cells. Virus protein synthesis was not required for the virus effect on bacterial invasiveness in the early infection phase, but it seems to be necessary in the late phase.

Coxsackie B1 virus did not replicate when microinjected into HEp-2 cells. Replication was assayed by production of infectious virus particles, synthesis of viral RNA, and lysis of cells. The same virus preparation initiated replication when it was inoculated into HEp-2 cells similarly to that by microinjected purified coxsackie B1 virus RNA.

Several hepatic peroxisome proliferators (HHPs) such as di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)-phthalate, clofibrate and tiadenol, induce morphological transformation of Syrian hamster embryo (SHE) cells in vitro. According to one hypothesis, the hepatocarcinogenic effect of HPPs is caused by an oxidative stress due to increased H2O2-production from the strongly induced peroxisomal beta-oxidation of fatty acids. Thus, increased transformation frequencies by HPPs should be obtained when catalase was inhibited by 3-amino-1,2,4-triazole (amitrole). However, co-exposure to HPPs and amitrole did not enhance the transformation frequencies for any of the HPPs. The sensitivity of SHE cells for oxidative agents was studied by using menadione and H2O2. Menadione only induced transformation at a toxic concentration, while H2O2 induced transformation at non-toxic concentrations. To study the generation of oxidative radicals in SHE cells, electron spin resonance was employed. No oxidative radical formation was detected in tiadenol- or DEHP-exposed SHE cells. When menadione or H2O2 were added during the measurements, oxidative radicals were found. A transmission electron microscopic study showed a small number of peroxisomes, and did not reveal any increase in the number of peroxisomes in clofibrate-treated SHE cells.

Several heavy metal salts [NiSO4, Cd(CH3COO)2, Pb(CH3COO)2, K2CrO4, CrCl3] were examined for their effects on intercellular communication in primary Syrian hamster embryo (SHE) cells and in the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-sensitive SHE cell line BPNi. Two exposure regimes were used: the standard regime where the exposure occurred after the cells had grown to confluence; and a non-standard regime where a high number of cells were seeded in a medium containing the metal salts. None of the chemicals were potent inhibitors of communication, i.e. effects were only found at concentrations that were non-compatible with long-term survival of the cells. NiSO4 inhibited communication in both regimes and in both cell types, but the effect was more pronounced for BPNi cells in the non-standard regime. K2CrO4 inhibited communication in the non-standard regime in both cell types, but significantly increased communication in the standard regime in BPNi cells. Similar effects were not found with CrCl3 or KCl. Thus, they were due to the properties of the CrO4(2-) ion. K2CrO4 upregulated communication in TPA-exposed BPNi cells. A high concentration of SO4(2-) did not influence the K2CrO4 inhibition in the non-standard regime, but it significantly inhibited the K2CrO4-induced increase in communication in the standard regime in BPNi cells. This suggests that K2CrO4 acts through two different mechanisms in the two exposure regimes. The CrO4(2-)-sensitive site that causes enhancement of communication in the standard regime is probably intracellular, while the site causing CrO4(2-) inhibition in the formation of gap junctional communication in the non-standard regime is most likely to be extracellular.

Okadaic acid is a potent non-phorbol ester mouse skin tumor promoter. Unlike the phorbol ester tumor promoters, okadaic acid is unable to promote the induction of morphological transformation in Syrian hamster embryo cell colonies. On the contrary, okadaic acid seems to counteract the effect of phorbol esters on transformation. Also unlike phorbol ester tumor promoters, okadaic acid does not inhibit intercellular communication, neither in primary hamster embryo cells, nor in the phorbol ester sensitive cell line BPNi. Furthermore, okadaic acid has no effect on the reoccurrence of communication following removal of 12-O-tetradecanoylphorbol-13-acetate.

The effects of the hepatic peroxisome proliferators (HPPs) clofibrate, di-(2-ethylhexyl)-phthalate (DEHP), mono-(2-ethylhexyl)phthalate (MEHP) and 2,4-dichlorophenoxy acetic acid (2,4-D) on the activities of some peroxisome-associated enzymes and marker enzymes for other organelles, have been studied in primary Syrian hamster embryo (SHE) cells and Wistar rat embryo (WRE) cells. The majority of the cells are fibroblast-like. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) was included as it has been suggested that it may act as a peroxisome proliferator. The specific activities of catalase, fatty acyl-CoA oxidase (FAO) and peroxisomal beta-oxidation were approximately 100-fold lower in the embryonic cells than in rat hepatocytes. Other peroxisome-associated oxidases were not detected. The dihydroxyacetone-phosphate acyltransferase (DHAPAT) activity was comparable to that in rat liver. Marker enzymes for other organelles had specific activities comparable to rat hepatocytes. Catalase was shown by digitonin titration to be contained in a peroxisome-like compartment in both SHE and WRE cells. Clofibrate, DEHP and MEHP increased the catalase activity, which might suggest peroxisome proliferation. However, the findings that FAO and peroxisomal beta-oxidation did not increase or only very slightly, argue against peroxisome proliferation. 2,4-D and TPA induced no or only a very slight increase in the catalase activity.

The effects of four isomers of dinitrotoluene (DNT) and technical DNT (a mixture of DNT isomers and other compounds, with 2,4-DNT as the major constituent) were studied in two short-term in vitro assays. None of the isomers or technical DNT induced an increase in morphological transformation of Syrian hamster embryo (SHE) cells. Four DNT metabolites (2,4-diaminotoluene, 2-amino-4-nitrotoluene, 2-amino-6-nitrotoluene, and 2,4-dinitobenzoic acid), representing different stages in reduction or oxidation of DNT isomers, were also negative for induction of morphological transformation. The DNT isomers were tested in an intercellular communication assay based on dye transfer. 2,4-DNT, 2,6-DNT, and technical DNT inhibited intercellular communication in the SHE cell line BPNi at toxic concentrations. This may be reminiscent of in vivo data showing promoting activity of these compound. 2,3-DNT and 3,4-DNT did not inhibit communication.

The abilities of the hepatic peroxisome proliferators (HPPs) clofibrate, di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)-phthalate (MEHP), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) and tiadenol to induce morphological transformation and to increase the catalase activity of Syrian hamster embryo (SHE) cells were studied. DEHP, MEHP, clofibrate and tiadenol induced morphological transformation of SHE cells and increased the catalase activity. DEHP was more potent than clofibrate and tiadenol in both inducing catalase and morphological transformation, while MEHP seemed more potent than DEHP in inducing catalase, but not morphological transformation, 2,4,5-T and 2,4-D did not induce morphological transformation, but 2,4,5-T was more potent than clofibrate in increasing the catalase activity. These results show that several HPPs induce morphological transformation of SHE cells and an increase in the catalase activity. There is, however, no direct connection between these two parameters, as seen from the results of 2,4,5-T. The tumor promoter TPA, and the metal salt nickel sulphate, induced morphological transformation of SHE cells without any appreciable increase in the catalase activity. These results further corroborate the dissociation between induction of morphological transformation and the increase in catalase activity.

The ability of different mineral fibres to induce morphological transformation of Syrian hamster embryo cells has been studied. Increased transformation frequencies were obtained in the presence of chrysotile, crocidolite, amosite, anthophyllite and the glass fibres (GF) 100, while no significant increase in transformation frequency was observed with GF 110 and TiO2. Chrysotile was the most potent of the fibres tested. GF 100 was more potent than crocidolite, amosite and anthophyllite. By comparing transformation frequency and toxicity, it could be concluded that induction of transformation is not caused by unspecific cytotoxic effects. In contrast to some earlier studies, no synergistic effect was observed between benzo[a]pyrene (BaP) and asbestos fibres. Adsorption of BaP to crocidolite fibres had no effect on the transformation frequency. Moreover, crocidolite was not able to promote the transformation of cells pre-exposed to BaP. Electron microscopy studies showed that the fibres were rapidly phagocytosed. Blebs were often formed on the cell surface and were most pronounced after crocidolite exposure. The blebs did not seem to be associated with the areas of physical interaction between the cells and the fibres, but were distributed throughout the cell surface.