Human T47D-ERß breast cancer cells with tetracycline-dependent ERß expression reflect ERα/ERß ratios in rat and human breast tissue.

T47D-ERß breast cancer cells with tetracycline-dependent ERß expression and constant ERα expression can be used to investigate effects of varying ERα/ERß ratios on estrogen-induced cellular responses. This study defines conditions at which ERα/ERß ratios in T47D-ERß cells best mimic ERα/ERß ratios in breast and other estrogen-sensitive tissues in vivo in rat as well as in human. Protein and mRNA levels of ERα and ERß were analyzed in T47D-ERß cells exposed to a range of tetracycline concentrations and compared to ERα and ERß levels found in breast, prostate, and uterus from rat and human origin. The ERα/ERß ratio in T47D-ERß cells exposed to >150ng/ml tetracycline is comparable to the ratio found in rat mammary gland and in human breast tissue. The ERα/ERß ratio of other estrogen-sensitive rat and human tissues can also be mimicked in T47D-ERß cells. The ERα/ERß ratio found in MCF-7 and native T47D breast cancer cell lines did not reflect ratios in analyzed rat and human tissues, which further supports the use of T47D-ERß cells as model for estrogen-responsive tissues. Using 17ß-estradiol and the T47D-ERß cells under the conditions defined to mimic various tissues it could be demonstrated how these different tissues vary in their proliferative response.

Fenamates: a novel class of reversible gap junction blockers.

The effect of fenamates on gap junctional intercellular communication was investigated in monolayers of normal rat kidney (NRK) fibroblasts and of SKHep1 cells overexpressing the gap junction protein connexin43 (Cx43). Using two different methods to study gap junctional intercellular communication, single electrode voltage-clamp step response measurements and dye microinjection, we show that fenamates are reversible blockers of Cx43-mediated intercellular communication. After adding fenamates to a confluent monolayer of electrically coupled NRK fibroblasts, the voltage step-induced capacitive current transient changed from a transient characteristic for charging multiple coupled cell capacitances to one characteristic for a single cell in isolation. The capacitance of completely uncoupled cells was 19.7 +/- 1.0 pF (mean +/- S.E.M.; n = 11). Junctional conductance between the patched cell and the surrounding cells in the monolayer changed from >140.7 +/- 9.6 nS (mean +/- S.E.M.; n = 14) to <1.4 +/- 0.4 nS (mean +/- S.E.M.; n = 11) after uncoupling. Electrical coupling could be restored to >51.8 +/- 4.2 nS (mean +/- S.E.M.; n = 11) by washout of the fenamates. Voltage-clamp step response measurements showed that the potency of fenamates in inhibiting electrical coupling decreases in the order meclofenamic acid > niflumic acid > flufenamic acid. The half-maximal concentration determined by dye-coupling experiments was 25 and 40 microM for meclofenamic acid and flufenamic acid, respectively. Inhibition of gap junctional communication by fenamates did not involve changes in intracellular calcium or pH, and was unrelated to protein kinase C activity or an inhibition of cyclooxygenase activity. Voltage-clamp step response measurements in confluent monolayers of SKHep1 cells that had been stably transfected with Cx43 revealed that fenamates are potent blockers of Cx43-mediated intercellular communication. In conclusion, fenamates represent a novel class of reversible gap junction blockers that can be used to study the role of Cx43-mediated gap junctional intercellular communication in biological processes.

Inhibition of intercellular communication and induction of ethoxyresorufin-O-deethylase activity by polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans in mouse hepa1c1c7 cells.

Inhibition of intercellular communication (IC) between hepa1c1c7 cells was used as a possible bioassay to predict tumor promoting potency of polyhalogenated aromatic hydrocarbons (PAHs). Relative potencies with regard to 2,3,7,8-TCDD to inhibit IC and to induce cytochrome P450IA1/2 (EROD) in these hepa1c1c7 cells were compared in order to investigate the possible role of the Ah receptor (AhR). For the PCDD/F and the co-planar PCB congeners relative potencies of both responses were within the same range. However, the mono-ortho PCBs, 2,3,3',4,4'-PeCB, 2,3,4,4',5-PeCB, 2,3',4,4',5-PeCB and 2,3,3',4,4',5,5'-HxCB showed a 30-1300 times higher potency to inhibit IC compared to EROD induction activity. These potency differences were even more pronounced for the di-ortho PCBs, 2,2',5,5'-TeCB and 2,2',3,3',4,4'-HxCB. The data presented here indicate that for IC inhibition by these non-planar PCBs a non-AhR mediated mechanism, with a different structure-activity relationship may be responsible. Given the high IC inhibition potency of mono- and di-ortho PCBs and their abundancy in environmental mixtures, the mono- and di-ortho PCBs may contribute for a major part to the total tumor promoting potency of complex mixtures relevant to human exposure. Using the traditional TEF values, these compounds do not account for much toxic potency in a mixture, which may imply that the tumor promotion potential is not covered by the commonly derived TEF values.

Species-specific antagonism of Ah receptor action by 2,2',5,5'-tetrachloro- and 2,2',3,3'4,4'-hexachlorobiphenyl.

Using recombinant cell lines showing Ah receptor-controlled expression of a luciferase reporter gene, the interaction of di-ortho-substitute polychlorinated biphenyls (PCBs) with Ah receptor agonists was studied. In the recombinant Hepa1c1c7 mouse hepatoma (H1L1.1c7) cells strong antagonistic interaction of 2,2',5,5'-tetrachlorobiphenyl (PCB52) with luciferase expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3',4,4'-tetrachlorobiphenyl (PCB77) was observed, and similarly, between 2,2',3,3',4,4'-hexachlorobiphenyl (PCB128) and PCB77. Accordingly, PCB52 was found to inhibit ethoxyresorufin-O-deethylase (EROD) induction by PCB77 in wild-type Hepa1c1c7 cells. In contrast, the antagonistic effect of PCB52 on TCDD-induced luciferase expression was only minor in recombinant guinea pig GPC16 colon adenocarcinoma (G16L1.1c8) and human HepG2 hepatoma (HG2L1.1c3) cells, and intermediate in recombinant H4IIE rat hepatoma (H4L1.1c4) cells. Gel retardation studies using a 32 P-labelled dioxin responsive element (DRE)-containing oligonucleotide, and ligand binding studies using [3H]TCDD, demonstrated that the species-specific antagonistic activity of PCB52 on Ah receptor-controlled luciferase expression is due to inhibition of Ah receptor ligand and DNA binding. We conclude, that Ah-mediated luciferase expression provides a useful tool to study the species specificity of Ah receptor (ant)agonists.

Inhibition of gap junctional intercellular communication between primary human smooth muscle cells by tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF alpha), a pleiotrophic cytokine present in atherosclerotic lesions, caused a dose-dependent and persistent reduction in gap junctional intercellular communication (GJIC) between primary human smooth muscle cells in vitro. A continuous presence of TNF alpha was required for this persistent inhibition. Pretreatment of smooth muscle cells with ascorbic acid, alpha-tocopherol or glutathione prevented this inhibition of GJIC by TNF alpha. The persistent blockage of GJIC by continuous exposure to TNF alpha suggests that TNF alpha may share some mechanistic similarities with exogenous tumor promoters. Furthermore, this reduction in GJIC by TNF alpha may provide an additional link between the processes of atherosclerosis and carcinogenesis. The protection afforded by antioxidant compounds suggests a role for active oxygen species in the promotion stage of atherosclerosis.

Inhibition of intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like PCBs in mouse hepatoma cells (Hepa1c1c7): involvement of the Ah receptor.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and the coplanar 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (3,3',4,4',5,5'-HCB) on intercellular communication (IC) were determined in order to investigate the in vitro tumor promoting potency of these compounds. 2,3,7,8-TCDD and the two coplanar PCBs tested caused a rapid (2 hr) and a sustained inhibition (48 hr) of IC in the mouse hepatoma cell line (Hepa1c1c7) to 20 and 50% of the unexposed control, respectively. Inhibition of IC was dose dependent with an EC50 range of about 50-100 pM for 2,3,7,8-TCDD, 2-5 nM for 3,3',4,4'-TCB, and 10-15 nM for 3,3',4,4',5,5'-HCB, respectively. A comparison of the IC inhibitory effects of 2,3,7,8-TCDD and PCBs with a well-known aryl hydrocarbon receptor (AhR)-mediated response, the induction of ethoxyresorufin O-deethylase (EROD) activity, in the same cells revealed EROD induction by 2,3,7,8-TCDD, 3,3',4,4'-TCB, and 3,3',4,4',5,5'-HCB with EC50 ranges of 100-200 pM, 20-70 nM, and 5-10 nM, respectively. The time course of IC inhibition was paralleled by EROD induction, although the time of onset of the response was earlier for IC (1 hr) than for EROD (2.5 hr). A role of the AhR in the inhibition of IC by 2,3,7,8-TCDD and PCBs was demonstrated by the lack of inhibition in AhR-defective Hepa1c1c7 cells. Transient inhibition of IC was observed in the mutant cells only at early time points (within 2 hr of exposure). These results and the observation that alpha-naphtoflavone (an AhR antagonist) greatly reduced the 2,3,7,8-TCDD-dependent sustained inhibition of IC strongly support a role of the AhR in the sustained inhibition of IC by these compounds. Furthermore, these data suggest that the mouse Hepa1c1c7 cells may be a good model in which to study in vitro tumor promoting capacity of dioxins, PCBs, and related compounds.

Effect of lipids and aldehydes on gap-junctional intercellular communication between human smooth muscle cells.

Inhibition of intercellular communication is an important feature in the tumour promotion phase of a multistage carcinogenesis model. In atherosclerosis inhibition of cell-cell communication by atherogenic compounds, e.g., low density lipoproteins (LDL), also seems to be important. For testing atherogenic compounds we used an atherosclerosis relevant cell type, namely human smooth muscle cells. In order to investigate which part of the LDL particle would be involved in inhibition of metabolic co-operation between human smooth muscle cells in culture we tested several fatty acids and their breakdown products, namely aldehydes. Unsaturated C-18 fatty acids markedly influenced gap-junctional intercellular communication (GJIC), whereas saturated (C18:0, C16:0) and unsaturated fatty acids with > 20 carbon atoms did not inhibit GJIC. In the case of oleic and elaidic acid, orientation seemed important; however, after exposure to palmitoleic and palmitelaidic acid no differences were found. The most potent inhibitor of GJIC was linoleic acid, which inhibited GJIC by 75%. No correlation was found between degrees of unsaturation and ability to inhibit GJIC. Of the tested aldehydes, hexanal, propanal, butanal and 4-hydroxynonenal did significantly inhibit GJIC, while pentanal had no effect. Since modification of LDL was shown to be important in order for LDL to inhibit GJIC, these results show that fatty acids and their oxidative breakdown products may be of importance for the inhibition of GJIC by LDL.

Effects of cholesterol and oxysterols on gap junctional communication between human smooth muscle cells.

Intercellular communication is considered to play an essential role in maintaining and controlling cell growth, cell differentiation and homeostasis. Cell-cell communication can be regulated by factors that influence gap junctional function. In this study it was demonstrated that cholesterol and oxidized cholesterol have the potential to modulate gap junctional communication between human smooth muscle cells in an opposite way. Cholesterol supplementation to human smooth muscle cells resulted in an increase of gap junctional communication up to 130% with regard to the control values. However, autooxidized cholesterol inhibited gap junctional communication more than 40%. Testing of several pure cholesterol oxidation derivates on gap junctional communication demonstrated that all of them were capable to inhibit intercellular communication in the order 25-hydroxycholesterol greater than cholestan-3 beta,5 alpha,6 beta-triol greater than 7-ketocholesterol greater than cholesterol 5,6 alpha-epoxide. The cell-cell communication-inhibiting potency of these oxysterols is in accordance with their atherogenic potency. This implies that cholesterol oxidation products, instead of pure cholesterol, can be promoting factors in the atherogenesis by influencing gap junctional communication between arterial smooth muscle cells, the target cells of atherosclerotic lesions.

In-vitro testing and the carcinogenic potential of several nitrosated indole compounds.

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.

Modulation of low-density lipoprotein-induced inhibition of intercellular communication by antioxidants and high-density lipoproteins.

In order to study the capacity of antioxidants and high-density lipoproteins (HDL) to modulate the effects of low-density lipoprotein (LDL) on intercellular communication, arterial smooth muscle cells and a dye transfer method were used. LDL, in contrast to HDL, inhibited the communication between arterial smooth muscle cells from human umbilical cord and thoracic aorta in a dose-dependent manner. LDL, which can be oxidized, as detected by the lipid-peroxidation assay and gel electrophoresis, did not influence cell-cell communication in the presence of the antioxidants butylated hydroxytoluene (BHT), alpha-tocopherol and glutathione. The results suggest that LDL must undergo oxidative modification before it can influence intercellular communication. Like antioxidants, HDL diminished the LDL-induced inhibition of cell-cell communication. This study suggests that the modulation of gap-junctional communication by the balance of HDL and LDL in plasma may influence atherogenesis.

Inhibition of intercellular communication in smooth muscle cells of humans and rats by low density lipoprotein, cigarette smoke condensate and TPA.

Inhibition by tumor promoting chemicals of intercellular communication via gap junctions may be important in carcinogenesis. In order to investigate the possible role of gap junctional intercellular communication in atherogenesis, we examined the effect of known inhibitors of intercellular communication, 12-O-tetradecanoylphorbol-13-acetate (TPA) and cigarette smoke condensate (CSC), and low density lipoproteins (LDL) and high density lipoproteins (HDL) on cellular communication in smooth muscle cells of human and rat by the microinjection-dye transfer technique. When lucifer yellow CH solution is injected into a cell, the average numbers of human and rat smooth muscle cells that become fluorescent is about 22 and 6, respectively. The tumor promoter (TPA) almost completely blocked gapjunctional communication between smooth muscle cells at 100 ng/ml after 4 h exposure. LDL and CSC were able to inhibit intercellular communication in human and rat cells in a dose-dependent manner up to 60%. LDL-pretreatment of human smooth muscle cells did not affect inhibition of intercellular communication, which suggests that this effect is mainly non-receptor mediated. HDL did not influence junctional communication. The results indicate that inhibition of intercellular communication may also contribute to the pathogenesis of atherosclerotic lesions, such as plaques.

Effects of cigarette smoke condensate and 12-O-tetradecanoylphorbol-13-acetate on gap junction structure and function in cultured cells.

The effects of cigarette smoke condensate (CSC) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on gap junction structure, quantity and function were investigated. Gap junction morphology was studied in rotary-shadowed freeze-fracture replicas of primary chick embryo hepatocytes. CSC (24 micrograms/ml) induced a strong decrease of gap junction areas; within 6 h the areas were reduced by greater than 60%. In the first 3 h of exposure, TPA (100 ng/ml) also reduced gap junction areas, but in the next 3 h a partial recovery was observed. Protoplasmic fracture face centre-to-centre particle spacings were used as a measure for gap junction coupling. CSC had a slow (although not significant) reducing effect on particle spacings, while TPA induced a reduction from 10.6 nm (control) to 10.0 nm within 3 h, indicating a reduction of coupling. Gap junctions were quantified in thin sections of cultured chick embryo hepatocytes, V79 fibroblasts, and co-cultivated hepatocytes and V79 cells. CSC did not influence gap junction numbers in any of these cultures, while TPA treatment caused a disappearance of gap junctions between hepatocytes and between hepatocytes and V79 cells in the first 12 h of cultivation. In the following 36 h a slow recovery could be observed. Gap junctions between V79 cells had already disappeared within 30 min. Metabolic co-operation between hepatocytes and hypoxanthine-guanine phosphoribosyltransferase-deficient V79 cells was quickly and continuously blocked by CSC over 27 h, whereas the phorbol ester induced a transient block. The dissimilar effects of these compounds on both gap junction structure and function indicate that they act via different mechanisms. The finding that CSC did not inhibit phorbol ester protein kinase C binding and did not activate this protein kinase in vitro supports this hypothesis.

The release of mutagens from airborne particles in the presence of physiological fluids.

Airborne particulates collected indoors in residences and outdoors were extracted by soxhlet extraction and sonication with methanol. In a comparative study in which mutagenic activity was evaluated in the Salmonella typhimurium reversion assay both soxhlet extraction and sonication proved to be suitable extraction methods. First, the residue, obtained by sonication of loaded filters with methanol followed by evaporation to dryness (tar), was sonicated with newborn calf serum and lung lavage fluid from pigs. All serum extracts of the tar were mutagenic to Salmonella typhimurium TA98, and contained direct- and indirect-acting mutagens. However, the mutagenic activity recovered by serum was only about half of the total mutagenic activity of the tar. The other part of the mutagenic activity remained in the tar. Lung lavage fluid was only able to remove 5-10% of direct-acting mutagens from the tar of all samples. About 20% of indirect-acting mutagens from indoor air were recovered in lung lavage fluid, while the lung lavage fluid extract from outdoor air did not show indirect mutagenic activity. Second, mutagenic activity recovered by direct sonication of the filters with physiological fluids was comparable with the recovery obtained by sonication of the tar. However, after sonication of the filter with lung lavage fluid hardly any mutagenic activity remained on the filter, whereas after sonication of the tar a clear mutagenic activity was observed in the non-soluble residue.

Effect of retinol and cigarette-smoke condensate on dye-coupled intercellular communication between hamster tracheal epithelial cells.

The dye-coupled intercellular communication across gap junctions in primary hamster tracheal epithelial cells has been studied in serum-free, hormone-supplemented medium. In the absence of vitamin A, non-cytotoxic concentrations of cigarette-smoke condensate (CSC) inhibited intercellular communication between tracheal epithelial cells in a concentration-dependent way. All-trans retinol and retinoic acid showed biphasic effects on intercellular communication depending on their concentration. Physiological concentrations of retinol and retinoic acid increased the dye-coupled transfer of Lucifer Yellow CH via gap junctions compared with the dimethylsulfoxide-treated tracheal epithelial cells. At pharmacological concentrations retinol slightly increased the intercellular communication in the first 2 h of the exposure period, whereas upon longer treatment times with retinol and retinoic acid, gap-junction-mediated intercellular communication was inhibited almost completely. When retinol was given to tracheal epithelial cells before exposure to CSC or simultaneously with CSC-exposure, retinol counteracted the inhibitory potential of CSC on intercellular communication. The results of the present study clearly indicate that both CSC and all-trans retinol influence the intercellular communication between primary hamster tracheal epithelial cells in serum-free, hormone-supplemented culture medium.