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W A Toscano

Publications and source records attributed to W A Toscano.

At least 19 recordsLinked to original sources
PubMed2001

Posttranscriptional silencing of cytochrome P4501A1 (CYP1A1) during zebrafish (Danio rerio) development.

Induction patterns of cytochrome P4501A1 (CYP1A1), an early biochemical marker of exposure to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) were investigated during zebrafish (Danio rerio) development. A zebrafish CYP1A1 cDNA fragment was cloned and used to detect CYP1A1 mRNA in embryos exposed to TCDD (1 or 10 nM). Induction of CYP1A1 activity was dependent on age and state of hatch. CYP1A1 mRNA was observed by 15 hr postfertilization. CYP1A1 protein and monooxygenase activity were not detected until 3 days postfertilization and after hatch, as determined by Western immunoblot analysis and ethoxyresorufin O-deethylase (EROD) activity, respectively. In contrast to embryos, concomitant induction of mRNA and activity was detected in juvenile zebrafish (3 days posthatch) after 6 hr of TCDD exposure. Asynchronous induction of CYP1A1 mRNA and activity during development may be a general regulatory mechanism, as similar ontogenetic expression of this gene was demonstrated in mouse embryos. To our knowledge, this is the first report of CYP1A1 posttranscriptional silencing during embryogenesis. Our data suggest that TCDD-mediated induction of CYP1A1 activity is regulated differentially in developing and mature systems.

Aging↗
PubMed2001

Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio).

We developed an inducible in vivo reporter system to examine expression of the aryl hydrocarbon receptor (AhR) during development in zebrafish (Danio rerio). AhR is a ligand-activated transcription factor that mediates the toxic actions of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Induction of cytochrome P4501A1 (CYP1A1) is an early biomarker of AhR activation. A 1905 base pair region of the human CYP1A1 promoter/enhancer region was regulated by AhR in zebrafish liver cells after exposure to TCDD (10 nM) in a transient transfection assay. This regulatory region was fused to the cDNA sequence encoding green fluorescent protein (GFP) of jellyfish (Aequorea victoria). Transgenic zebrafish were generated to express this AhR-regulated GFP construct. Injected fish exposed to TCDD exhibited induction of GFP in the eye, nose, and vertebrae of zebrafish embryos (48 and 72 hr after fertilization) compared to vehicle controls (DMSO), which did not express GFP. To investigate whether AhR-regulated GFP expression correlated with sites of TCDD toxicity, we exposed wild-type zebrafish to DMSO or TCDD and examined them for morphologic abnormalities. By 5 days after fertilization, TCDD-exposed fish exhibited gross dysmorphogenesis in cranio-facial and vertebral development.

Animals↗
PubMed1999

Estrogen receptor reduces CYP1A1 induction in cultured human endometrial cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exerts its toxic action via the aryl hydrocarbon (Ah) receptor, which induces a battery of xenobiotic-metabolizing enzymes, including the cytochrome P450 isozyme, CYP1A1. TCDD-induced 7-ethoxycoumarin-O-deethylase activity was reduced 75% in cultured human endometrial ECC-1 cells exposed to various concentrations of 17beta-estradiol for up to 72 h, with a half-maximal effective concentration (EC50) of 0.9 nM. Reduced enzyme activity was correlated with decreased CYP1A1 mRNA levels, and transcription. Exposure to TCDD plus 17beta-estradiol also reduced CYP1A1 activity in MCF-7 breast cancer cells but not in Hep-3B human liver cells or HuE primary human keratinocytes, suggesting that the effect was specific to estrogen-regulated cells. Estrogen receptor antagonists 4-hydroxytamoxifen and 7alpha-[9-(4,4, 5,5,5-pentafluoro-pentylsulfinyl)nonyl]estra-1,3,5(10)-tr iene3, 17beta-diol restored TCDD-induced CYP1A1 transcription, steady-state mRNA levels, and enzymatic activity in ECC-1 cells. Gel mobility shift assay showed that 17beta-estradiol had little effect on Ah receptor binding to its DNA-responsive element. 17beta-Estradiol did not alter the induction of another Ah receptor-regulated gene, CYP1B1, suggesting that altered Ah receptor binding to DNA does not mediate reduced CYP1A1 transcription. Transfecting ECC-1 cells with a general transcription factor involved in CYP1A1 induction, nuclear factor-1, reversed 17beta-estradiol antagonism of dioxin induced-CYP1A1. The data suggest that 17beta-estradiol reduced CYP1A1 expression at the transcriptional level by squelching available nuclear factor-1, a transcription factor that interacts with both Ah and estrogen receptors.

Cell Line↗
PubMed1999

ECC-1 human endometrial cells as a model system to study dioxin disruption of steroid hormone function.

ECC-1, an established epithelial cell line derived from an adenocarcinoma of human endometrial lining, was examined for growth optimization, steroid hormone receptor- and Ah receptor content, and dioxin modulation of estrogen receptor function. Proliferation of ECC-1 cells was accelerated by growth on a lethally irradiated feeder layer of murine 3T3 fibroblasts. Immunoblot analysis demonstrated the presence of Ah receptor an intracellular protein that binds and regulates the toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Ah receptor was functional in these cells as assessed by concentration and kinetic patterns of CYP1A1-mediated 7-ethoxycoumarin O-deethylase (ECOD) induction. The half-maximal effective concentration (EC50) for TCDD was 0.2 nM, and maximal activity appeared after 24-h exposure. A limited structure-activity examination of ECOD activity provided additional evidence for Ah receptor involvement. Competitive binding assays were performed to examine kinetic parameters for estrogen, progesterone, and glucocorticoid receptors. Binding parameters of dissociation constant (Kd) and number of binding sites (Bmax) derived from Scatchard analysis were: estrogen, Kd = 0.67 nM; Bmax = 321 fmol/mg cytosolic protein; progesterone, Kd = 1.31 nM; Bmax = 258 fmol/mg cytosolic protein; dexamethasone, Kd = 1.75 nM, Bmax = 128 fmol/mg cytosolic protein. Exposure of ECC-1 cells to TCDD reduced the estrogen receptor level by 40% without affecting the Kd value, and reduced estrogen receptor-mediated transcription by 50% assessed by transient transfection of an estrogen-responsive reporter plasmid. These data suggest that the ECC-1 cell line is a useful model system for examining the action of dioxin in human endometrial tissue. Both the estrogen receptor and Ah receptor have been implicated in diseases of the endometrium, and examining their interactions may elucidate mechanisms of uterine disease etiology, as well as potential targets for disease prevention.

Cell Division↗
PubMed1998

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters retinoic acid receptor function in human keratinocytes.

Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on all-trans-retinoic acid (trans-RA) binding to retinoic acid receptors (RARs) in cultured human keratinocytes (SCC-12F) was investigated. TCDD and trans-RA elicited opposing actions on the production of biologically active TGF-beta. TCDD exposure caused concentration- and time-dependent decreases in trans-RA binding to SCC-12F RARs. The apparent half-maximal effective TCDD concentration = 1 nM. TCDD exerts its action via the aryl hydrocarbon receptor (AhR). TCDBF, a partial AhR agonist, reduced trans-RA binding, indicating AhR involvement (control = 0.33; TCDBF = 0.22; TCDD = 0.142 pmol trans-RA bound/mg nuclear protein). The dissociation constant (Kd) calculated from Eadie-Hofstee analysis of equilibrium binding for trans-RA was 0.13 nM in both TCDD-exposed and control cultures. Approximately half of the trans-RA binding sites were lost in TCDD-exposed cells (control = 0.195; TCDD = 0.108 pmol trans-RA bound/mg protein). The data suggest TCDD may exert its toxic action in human keratinocytes by directly modulating RAR action.

Animals↗
PubMed1997

Synergistic responses of steroidal estrogens in vitro (yeast) and in vivo (turtles).

Many environmental agents exert estrogenic activity. Previous studies from our laboratories demonstrated that certain combinations of environmental estrogens (i) reverse the sex of male turtle embryos in a synergistic manner (Bergeron et al., (1994) Environ. Hlth Perspect. 102, 780-782), and (ii) synergistically transactivate the human estrogen receptor (hER) in yeast and mammalian cells (Arnold et al., (1996) Science 272, 1489-1492). Because our findings with synthetic estrogens suggested that combinations of naturally-occurring steroidal estrogens might also produce synergistic activity of the ER, we used the same model systems to measure the activity of combinations of steroidal estrogens. The activity of combinations of estrone, estradiol-17beta or estradiol-17alpha in yeast strains expressing hER was synergistic at submaximal concentrations of both estrogenic compounds. However, synergy was not observed with mixtures of estrogens when the concentration of one of the estrogens alone was maximally active in yeast. Ligand-binding assays in yeast performed with various radiolabeled estrogens suggested that multiple estrogens may interact with the receptor. The estrogen-dependent process of sex-reversal of turtle embryos incubated at a male-producing temperature was used to determine whether steroidal estrogens also had synergistic activity in vivo. In this instance, a combination of estriol and estradiol-17beta was effective in reversing the gonadal sex of turtle embryos from males to females in a synergistic manner. Our results suggest that the synergy of some combinations of estrogens, synthetic or steroidal, may play a role in the estrogen-dependent process of sexual development in certain species.

Animals↗
PubMed1995

Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The action of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on transcription and steady-state mRNA levels, protein expression, and enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured human keratinocytes was examined. GAPDH transcription increased as a function of time of exposure to TCDD, reaching a maximum of 23-fold after 8 days. Northern blot analysis showed a 4-fold increase in mRNA after 8 days exposure. Protein levels were increased 4-fold, and dehydrogenase activity by 5-fold, under the same exposure conditions. GAPDH is commonly thought of as a constitutive housekeeping gene and is often used as a loading control for northern blot analysis. Our data suggest that caution should be used when using GAPDH as an RNA control, especially in studies examining cell proliferation and carcinogenesis.

Blotting, Northern↗
PubMed1991

Dioxin induces transforming growth factor-alpha in human keratinocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental toxicant, is a tumor promoter that induces hyperplasia in epithelial cells. Exposure of cultured human keratinocytes to TCDD, resulted in a time-dependent dioxin-specific Ah receptor-mediated release of transforming growth factor-alpha (TGF-alpha) into the culture medium. Cultures exposed to TCDD showed a rate of TGF-alpha secretion into the medium of about 30 fmol/ml/day, as well as a 3- to 6-fold increase in TGF-alpha mRNA expression. Increased production of TGF-alpha in human keratinocytes exposed to TCDD demonstrates a modulation of autocrine regulation in those cells. These results suggest that induction of TGF-alpha could be an important part of the mechanism of dioxin-mediated toxicity and tumor promotion.

Blotting, Northern↗
PubMed1991

Suppression of phorbol ester-enhanced radiation-induced malignancy in vitro by protease inhibitors is independent of protein kinase C.

X-irradiation and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act in a synergistic manner to increase the yield of transformed C3H10T1/2 cells in vitro. TPA modulated both translocation from the cytosol to the plasma membrane, and down regulation of protein kinase C (PKC) after prolonged (48 h) TPA exposure. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), antipain, and soybean-derived Bowman-Birk inhibitor, protease inhibitors that suppress transformation of C3H10T1/2 cells, had no effect on these TPA-mediated alterations of PKC activity, suggesting that protease inhibitors suppress TPA-stimulated promotion in vitro via a PKC-independent pathway. Several experiments were performed to determine whether non-toxic concentrations of the PKC inhibitors, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), TPCK, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), or 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine (H-7), modulated the movement of cells from a quiescent state into the cell cycle. TPCK and the combination of H-7 and W-7 lowered DNA synthesis when cells were stimulated to divide by TPA. Because other protease inhibitors that slow transformation in vitro did not have the same suppressive effect on DNA synthesis, the inhibitory pathway that suppresses carcinogenic activity is likely to be different from the suppression of DNA synthesis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Human glutathione S-transferase deficiency as a marker of susceptibility to epoxide-induced cytogenetic damage.

The identification of genetic traits that predispose individuals to environmentally induced cancers is one of the most important problems in cancer risk assessment. Genetic deficiency in the mu-isozyme of the glutathione (GSH) S-transferases (EC 2.5.1.18) has recently been associated with increased lung cancer risk. To test whether this association could arise from a metabolically mediated sensitivity to mutagenic substrates, cytogenetic damage in lymphocytes from 21 isozyme-deficient and 24 nondeficient individuals was induced. Cells were treated with trans-stilbene oxide, an excellent substrate for GSH S-transferase mu, or cis-stilbene oxide, a poor substrate for the isozyme. Sister chromatid exchange induction was measured as an indicator of cytogenetic damage. A trimodal distribution of trans-stilbene oxide-induced sister chromatid exchanges was observed in the population, including resistant, moderate, and highly sensitive groups. Glutathione S-transferase mu deficiency was associated with both moderate and high sensitivity to trans-stilbene oxide-induced damage but had no effect on cis-stilbene oxide-induced sister chromatid exchange. The results indicate that GSH S-transferase mu, a proposed marker of cancer susceptibility, is also a marker of susceptibility to the induction of cytogenetic damage by a certain class of mutagens. The differential effects of the cis- and trans-isomers of stilbene oxide illustrate that the stereoselectivity of GSH S-transferase mu toward various alkene epoxide substrates can be an important factor affecting individual sensitivity to DNA-damaging epoxides.

Adult↗
PubMed1988

Modulation of adenylate cyclase in human keratinocytes by protein kinase C.

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.

Adenosine Diphosphate Ribose↗
PubMed1987

Calmodulin-mediated adenylate cyclase from mammalian sperm.

Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.

Adenylyl Cyclases↗
PubMed1986

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates epidermal growth factor (EGF) binding to basal cells from a human keratinocyte cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates the proliferation, differentiation, or both, of epidermal keratinocytes in vivo and in culture. The growth of epidermal cells in culture is regulated by several biochemical mediators including epidermal growth factor (EGF). In this report the actions of TCDD on EGF binding in a basal cell population from a human keratinocyte cell line were examined. TCDD decreased the specific binding of 125I-EGF to basal cells by 40% within 96 hr. This reduction in EGF binding could not be attributed to changes in the state of differentiation as assessed by cell size and morphology, and cornified envelope competence, a marker of terminal differentiation. Modulation of EGF binding by TCDD was concentration-dependent (EC50 = 1 nM) and stereospecific, suggesting involvement of the Ah receptor. Scatchard analysis of EGF binding to the basal cells indicated a single class of high-affinity sites in both control (Kd = 0.14 nM) and treated (Kd = 0.11 nM) cultures and confirmed a decrease in the number of these sites in response to TCDD. The reduction in EGF binding correlated with a decrease in EGF-stimulated DNA synthesis and cell proliferation. Comparison of differentiation-competent squamous cell carcinoma (SCC) lines treated with TCDD supported an association between modulation of EGF binding and enhanced differentiation. The data indicate that basal cells are a target for TCDD. We propose that the modulation of EGF binding in basal keratinocytes by TCDD is one of the critical regulatory events resulting in enhanced differentiation.

Calcium↗
PubMed1985

Purification and properties of calmodulin from adrenal cortex.

Calmodulin (CaM), a multifunctional calcium binding protein with no known enzymatic activity, has been purified to homogeneity from bovine adrenal cortex. The purification included anion exchange on DE-52 cellulose, ammonium sulfate precipitation, and separation by molecular sieving on Sephadex G-150. The yield of CaM from 900 g of whole adrenal was 150 mg. Adrenocortical CaM showed a molecular weight of 18,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 4.1, and demonstrated a characteristic shift in mobility on polyacrylamide gels in the presence of calcium. The spectral properties of adrenocortical CaM differed slightly from those of CaM isolated from bovine brain. Minor differences were observed in peptide maps and amino acid composition between adrenocortical and brain CaM, but adrenocortical CaM contained a single trimethyl-lysine residue characteristic of all mammalian forms of CaM isolated to date. Adrenocortical CaM is biologically active in the stimulation of activator-deficient phosphodiesterase, and showed a half-maximal effective concentration (EC50) of 3 nM for stimulation of adenylate cyclase from Bordetella pertussis.

Adrenal Cortex↗
PubMed1985

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1).

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1985

Regulation of epidermal growth factor binding in a human keratinocyte cell line by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) decreased the binding of epidermal growth factor (EGF) by the human keratinocyte cell line SCC-12F. This response was concentration dependent (half-maximal effective concentration, EC50 = 1.8 nM) and stereospecific. Scatchard analysis of EGF binding indicated that treatment with TCDD resulted in a loss of high-affinity (Kd = 0.28 nM) binding sites. This loss was accompanied by a concomitant inhibition of EGF-stimulated DNA synthesis. The kinetics for the decrease of EGF binding by TCDD and benzo[a]pyrene (BP) were compared. Inhibition of EGF binding by BP was maximal by 24 hr, with 90% recovery of EGF binding apparent by 48 hr. In contrast, TCDD treatment for 72 hr was required to produce maximal inhibition, and no recovery was evident up to 10 day after removal of TCDD from the growth medium. The data indicate that modulation of EGF binding by TCDD was mediated by the Ah receptor. Subsequent cellular responses, for example, inhibition of EGF-stimulated DNA synthesis, may be important in the expression of altered differentiation patterns observed in human epidermal keratinocytes exposed to TCDD.

Cell Line↗