Response of preosteoblasts to thermal stress conditioning and osteoinductive growth factors

Conditioning protocols involving mechanical stress independently or with chemical cues such as growth factors (GFs) possess significant potential to enhance bone regeneration. However, utilization of thermal stress conditioning alone or with GFs for bone therapy has been under-investigated. In this study, a preosteoblast cell line (MC3T3-E1) was exposed to treatment with water bath heating (44°C, 4 and 8 min) and osteoinductive GFs (bone morphogenetic protein-2 and transforming growth factor-β1) individually or in combination to investigate whether these stimuli could promote induction of bone-related markers, an angiogenic factor, and heat shock proteins (HSPs). Cells remained viable when heating durations were less than 20 min at 40oC, 16 min at 42oC, and 10 min at 44oC. Increasing heating duration at 44°C, promoted gene expression of HSPs, osteocalcin (OCN), and osteopontin (OPN) at 8 h post-heating (PH). Heating in combination with GFs caused the greatest gene induction of osteoprotegerin (OPG; 6.9- and 1.6-fold induction compared to sham-treated and GF only treated groups, respectively) and vascular endothelial growth factor (VEGF; 16.0- and 1.6-fold compared to sham and GF-only treated groups, respectively) at 8 h PH. Both heating and GFs independently suppressed the matrix metalloproteinase-9 (MMP-9) gene. GF treatment caused a more significant decrease in MMP-9 protein secretion to non-detectable levels compared to heating alone at 72 h PH. Secretion of OCN, OPN, and OPG increased with the addition of GFs but diminished with heating as measured by ELISA at 72 h PH. These results suggest that conditioning protocols utilizing heating and GFs individually or in combination can induce HSPs, bone-related proteins, and VEGF while also causing downregulation of osteoclastic activity, potentially providing a promising bone therapeutic strategy.     

Dexamethasone increases αvβ3 integrin expression and affinity through a calcineurin/NFAT pathway

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvβ3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvβ3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvβ3 integrin was the result of an increase in the expression of the β3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p < 0.04) in β3 integrin mRNA by day 2 compared to control and remained elevated for 6 days of treatment and then an additional 10 days once the DEX was removed. The increase in β3 integrin mRNA levels required only 1 day of DEX treatment to increase levels for 4 days in the absence of DEX. In contrast, DEX did not alter β1 integrin mRNA or protein levels. The DEX-induced upregulation of β3 integrin mRNA was partly due to an increase in its half-life to 60.7 h from 22.5 h in control cultures (p < 0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in β3 integrin mRNA. In summary, the DEX-induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and the upregulation of the β3 integrin subunit through the calcineurin/NFAT pathway.     

Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Fivetrade mark cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Fivetrade mark cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Fivetrade mark cell cultures, but not in Sf-9 cell cultures. In addition, High Fivetrade mark cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Fivetrade mark cell cultures. It was also found that the medium had a significant effect on High Fivetrade mark cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.     

Modulation of sulfated proteoglycan synthesis and collagen gene expression by chondrocytes grown in the presence of bFGF alone or combined with IGF1

Prepubertal rabbit epiphyseal chondrocytes were grown in high density primary culture for 3 d. They were then incubated for 3 additional d in serum-free culture medium to which bFGF (1-50 ng/ml) was added. During the last 24 h incubation period, either IGF1 (1-80 ng/ml) or Insulin (1-5 micrograms/ml) was added to the culture medium. Chondrocyte DNA was significantly augmented with the increasing concentration of bFGF used, thus confirming its mitogenic effect on chondrocytes. On the other hand, bFGF was also shown to modulate the phenotypic expression of the chondrocytes. The 35S-sulfate incorporation into newly synthesized proteoglycans by the cultured cells decreased in a dose-dependent manner with bFGF concentration used. In addition, chondrocyte collagen gene expression was also shown to be modulated by bFGF. Total RNA extracted from the cultured cells was analyzed by dot blot and Northern blot with cDNA probes encoding for alpha 1 II and alpha 1 I procollagen chains. A significant lower level of type II collagen mRNA, the marker of chondrocytic phenotype, was observed when cells were grown in the presence of bFGF while the level of type I mRNA remained unchanged. When IGF1 or a high concentration of insulin was added to the cells during the last 24 h of incubation with bFGF, sulfated proteoglycan synthesis, as well as collagen type II mRNA level, were significantly stimulated when compared with chondrocytes incubated with bFGF alone. In conclusion, in the present experimental conditions, bFGF appears to be a growth promoting agent for chondrocytes in vitro with dedifferentiating action on chondrocyte phenotype. IGF1 or insulin used at a high concentration can prevent the dedifferentiating effect of bFGF without inhibiting its stimulating effect on chondrocyte DNA synthesis.     

Matrix metalloproteinase 2 activity decreases in human periodontal ligament fibroblast cultures submitted to simulated orthodontic force

Orthodontic force compresses the periodontal ligament promoting the expression of pro-inflammatory mediators and matrix metalloproteinases responsible for tooth movement. The extent in time while periodontal cells are being treated and the increment in the amount of mechanical stress caused by the orthodontic force is thought to regulate the levels of metalloproteinases in the periodontal tissue. To study the possible regulation in the activity of metalloproteinases 2, 3, 7, 9, and 10 by simulated orthodontic force, human periodontal ligament fibroblast cultures were centrifuged (141×g) for 30, 60, 90, and 120 min, simulating the orthodontic force. Cell viability, protein quantification, and activity of metalloproteinases by zymography were evaluated at 24, 48, and 72 h after centrifugation in both cell lysates and growth medium. The activity of the 72-kDa matrix metalloproteinase 2 was decreased at 24 h regardless of the duration of centrifugation and at 48 h in cells centrifuged for 30 min only. Decrease in the amount of total protein in lysates was seen at 48 and 72 h with no change in cell viability. The data seem to indicate that the amount of mechanical stress regulates the levels of secreted matrix metalloproteinase 2. In addition, the centrifugation as a model for simulated orthodontic force may be used as a simple and reliable method to study the role played by matrix metalloproteinases in periodontal ligament when submitted to mechanical force as occurring during tooth movement.     

Short-term alterations in hippocampal glutamate transport system caused by one-single neonatal seizure episode: implications on behavioral performance in adulthood

Impairment in the activity and expression of glutamate transporters has been found in experimental models of epilepsy in adult animals. However, there are few studies investigating alterations on glutamate transporters caused by epilepsy in newborn animals, especially in the early periods after seizures. In this study, alterations in the hippocampal glutamate transporters activity and immunocontent were investigated in neonatal rats (7 days old) submitted to kainate-induced seizures model. Glutamate uptake, glutamate transporters (GLT-1, GLAST, EAAC1) and glutamine synthetase (GS) were assessed in hippocampal slices obtained 12 h, 24 h, 48 h, 72 h and 60 days after seizures. Immunoreactivity for hippocampal GFAP, NeuN and DAPI were assessed 24 h after seizure. Behavioral analysis (elevated-plus maze and inhibitory avoidance task) was also investigated in the adult animals (60 days old). The decrease on glutamate uptake was observed in hippocampal slices obtained 24 h after seizures. The immunocontent of GLT-1 increased at 12 h and decreased at 24 h (+62% and −20%, respectively), while GLAST increased up to 48 h after seizures. No alterations were observed for EAAC1 and GS. It should be mentioned that there were no long-term changes in tested glutamate transporters at 60 days after kainate treatment. GFAP immunoreactivity increased in all hippocampal subfields (CA1, CA3 and dentate gyrus) with no alterations in NeuN and DAPI staining. In the adulthood, kainate-treated rats showed anxiety-related behavior and lower performance in the inhibitory avoidance task. Our findings indicate that acute modifications on hippocampal glutamate transporters triggered by a single convulsive event in early life may play a role in the behavioral alterations observed in adulthood.     

Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: A step towards exploring a potential use of matrix-induced cell therapy

Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and ‘chondrocytes-fibrin’ construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM + 10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM + 2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM + 10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs > FD2GFs > FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and ‘chondrocytes-fibrin’ constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, ‘chondrocytes-fibrin’ constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality ‘chondrocytes-fibrin’ constructs which may have potential use of matrix-induced cell implantation.     

Effects of leptin and adiponectin on proliferation and protein metabolism of porcine myoblasts

The aim of this study was to show the abundance of leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) and to determine the direct effects of leptin and adiponectin on the in vitro growth of porcine skeletal muscle cells. ADIPOR1 and ADIPOR2 were abundant at mRNA and protein level in proliferating and differentiating myoblast cultures derived from semimembranosus and semitendinosus muscles of newborn piglets, whereas LEPR expression was close to the detection limit. Adiponectin (10, 20, 40 μg/ml) attenuated the proliferation of porcine myoblasts, measured as [(3)H]-thymidine incorporation and real-time monitoring of the cells in response to 24- and 48-h exposure, in a dose-dependent manner. This effect resulted from suppressed basic fibroblast growth factor (bFGF)-mediated stimulation of DNA synthesis in serum-free medium (SFM) containing bFGF. No effects of leptin (5, 10, 20, 40, 80 ng/ml) on myoblast proliferation in SFM were detectable. Neither leptin nor adiponectin altered protein synthesis and degradation in differentiating porcine myoblasts cultured in SFM. The results on receptor abundance suggest that porcine skeletal muscle cells may be sensitive to adiponectin and leptin. However, except via inhibitory interaction of adiponectin with bFGF, these adipokines appear not to affect in vitro proliferation and protein metabolism of porcine muscle cells directly under serum-free culture conditions.     

Effects of testosterone and 2-hydroxyflutamide on progesterone receptor expression in porcine ovarian follicles in vitro

The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T + 2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6–8 mm in diameter) were cultured for 48 h. Experimental cultures were carried out with the addition of T (10^?7 M), 2-Hf (1.7 × 10^?4 M) or both T and 2-Hf for the last 24 h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6–8 mm in diameter) were cultured for 24 h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P₄) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P₄ secretion, but simultaneous addition of 2-Hf and T increased P₄ secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.     

Autoradiographic studies of rat astroglial cell proliferation in vitro with and without treatment with basic fibroblast growth factor

Abstract. Using specific autoradiographic methods, cell cycle parameters of untreated and basic fibroblast growth factor (bFGF)-treated astroglial cells from newborn rats grown in primary culture were directly measured. The mode of proliferation was also analysed. In untreated cultures, S phase duration (T_s= 6.9–13.1 h) and cell cycle time (T_c= 10–18 h) can be modified by about a factor of 2 depending on the culture conditions (serum-supplemented or defined medium, thyroid hormone concentration). However, growth fraction (GF = 0.15) and the ratio T_s/T_c remain stable. With increasing days in vitro (DIV) (DIV 7-DIV 20), T_s (7.8–10.6 h) and T_c (10–21 h) are prolonged and GF (0.14–0.06) decreases, probably due to cell maturation. In general, astroglial cells proliferate exponentially with a GF < 1, but stop proliferating about 30–36 h after the last feeding, probably caused by exhaustion of the medium. However, after refeeding they continue to proliferate. As opposed to in vivo , no transition of non-proliferating cells into the GF occurs. After addition of bFGF, GF increases (e.g. GF at DIV 7 = 0.43), but T_s and T_c are not influenced at DIV 7 and 12. At DIV 20, bFGF additionally shortens T_s and T_c, thereby producing values of T_s, T_c and GF like 'younger' cultures. However, the revitalizing effect on 'mature' cells is only transitory. In general, bFGF leads to a single re-entry of G_o cells into the GF. Thereafter, bFGF does not affect the mode of proliferation.     

Effect of Some Growth Factors on Tissue Transglutaminase Overexpression Induced by β-Amyloid in Olfactory Ensheathing Cells

Herein, we assessed in a particular glial cell type, called olfactory ensheathing cells (OECs), the effect of some growth factors (GFs) on tissue transglutaminase (TG2) overexpression induced by amyloid-beta (Aβ) with native full-length peptide 1–42 or by fragments, 25–35 or 35–25, as control. Previously, we demonstrated that TG2 overexpression induced by some stressors was down-regulated by GFs exposure in OECs. To monitor cell viability, an MTT test was used, while TG2 expression was examined using immunocytochemical and Western blot analysis. We also considered the involvement of the TG2-mediated apoptotic pathway. Vimentin expression was evaluated as well. Reactive oxygen species and reduced glutathione levels were utilized to test the oxidative intracellular status. Lactate dehydrogenase released into the medium, as a marker of necrotic cell death, was evaluated. We found that in OECs exposed to Aβ(1–42) or Aβ(25–35) for 24 h, TG2 expression increased, and we observed that the protein appeared prevalently localized in the cytosol. The pre-treatment with GFs, basic fibroblast growth factor (bFGF) or glial-derived neurotrophic factor (GDNF), down-regulated the TG2 level, which was prevalently limited to the nuclear compartment. Vimentin expression and caspase cleavage showed a significant enhancement in Aβ(1–42) and Aβ(25–35) exposed cells. The pre-treatment with bFGF or GDNF was able to restore the levels of the proteins to control values, and the intracellular oxidative status modified by the exposure to Aβ(1–42) or Aβ(25–35). Our data suggest that both bFGF or GDNF could be an innovative mechanism to contrast TG2 expression, which plays a key role in Alzheimer’s disease.     

Glial shape and cytoskeletal protein synthesis

We investigated whether the shape of astroglial derived cells influences the expression of cytoskeletal proteins. In reaggregating cultures GFAP, vimentin and actin synthesis was approximately 52%, 50% and 37% the level found in monolayer cultures, respectively. Monolayer cultures consisted of polygonal shaped cells adhering to plastic, while reaggregating cultures were comprised of round cells growing in a suspension like culture. Additionally, human glioma cells induced to grow as round cells on poly-2-hydroxyethyl methacrylate (polyhema) coated plastic exhibited a level of GFAP synthesis that was approximately 20% the level displayed by polygonal shaped cells grown on uncoated plastic. Glioma cells initially grown on a polyhema surface and replated onto uncoated plastic were capable of reinitiating GFAP synthesis. Thus, aterations in the synthesis of GFAP and other cytoskeletal proteins can occur when astrocytes change their shape.     

Effect of Acetylcholine Precursors on Proliferation and Differentiation of Astroglial Cells in Primary Cultures

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5′-diphosphocholine (CDP-choline) and α-glyceril-phosphorylcholine (α-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 μM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 μM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 μM α-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 μM choline or α-GPC, whereas in 24 h 1 μM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 μM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 μM α-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.     

Effects of basic fibroblast growth factor on endothelial cells under conditions of simulated microgravity

Fibroblast growth factors interact with appropriate endothelial cell (EC) surface receptors and initiate intracellular signal cascades, which participate in modulating blood vessel growth. EC, upon exposure to basic fibroblast growth factors (bFGFs) undergo profound functional alterations, which depend on their actual sensitivity and involve gene expression and de novo protein synthesis. We investigated the effects of bFGF on signaling pathways of EA.hy926 cells in different environments. EC were cultured under normal gravity (1 g) and simulated microgravity (micro g) using a three-dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin-1 (ET-1) and TGF-beta(1) expression. Microgravity reduced eNOS mRNA within 24 h. Moreover, a six- to eightfold higher amount of IL-6 and IL-8 was secreted within 24 h micro g. In addition, microgravity induced a duplication of NF-kappaB p50, while p65 was quadrupled. At 1 g, bFGF application (4 h) reduced ET-1, TGF-beta(1) and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk-1, Flt-1, the release of IL-6, IL-8, and TGF-beta(1). Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h micro g, bFGF decreased TGF-beta(1), eNOS, and ET-1 gene expression. After 24 h micro g, bFGF elevated fibronectin, Flk-1 and Flt-1 protein, and reduced IL-6 and IL-8 compared with vehicle treated micro g cultures. In micro g, bFGF enhanced NF-KappaB p50 by 50%, Bax by 25% and attenuated p65, activation of caspase-3 and annexin V-positive cells. bFGF differently changes intracellular signals in ECs depending whether it is applied under microgravity or normal gravity conditions. In microgravity, bFGF contributes to protect the EC from apoptosis.     

Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro

Summary Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 × 10⁶ cells/cm² in plastic culture dishes precoated with trout skin extract (7.6 μg skin protein/cm²) to facilitate cell attachment were maintained at 16° C. Cells were treated with DEX (10⁻⁹ to 10⁻⁷ M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10⁻⁸ to 10⁻⁷ M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10⁻⁸ to 10⁻⁷ M). DEX at 10⁻⁹ M was ineffective. Concomitant addition of 10⁻⁶ M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10⁻⁷ M DEX abolished the DEX effect. RU486 at 10⁻⁸ M was ineffective. Spironolactone (10⁻⁸ to 10⁻⁶ M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10⁻⁶ M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).