Biomedical subjects

S Safe

Publications and source records attributed to S Safe.

At least 91 records · Page 5Linked to original sources

PubMed1996

Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells.

Indole-3-carbinol (I3C) is a major component of Brassica vegetables, and diindolylmethane (DIM) is the major acid-catalyzed condensation product derived from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor with relatively low affinity. In Ah-responsive T47D human breast cancer cells, I3C and DIM did not induce significantly CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as 125 or 31 microM, respectively. A 1 nM concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells, and cotreatment with TCDD plus different concentrations of I3C (1-125 microM) or DIM (1-31 microM) resulted in a > 90% decrease in the induced response at the highest concentration of I3C or DIM. I3C or DIM also partially inhibited (< 50%) induction of CYP1A1 mRNA levels by TCDD and reporter gene activity, using an Ah-responsive plasmid construct in transient transfection assays. In T47D cells cotreated with 5 nM [3H]TCDD alone or in combination with 250 microM I3C or 31 microM DIM, there was a 37 and 73% decrease, respectively, in formation of the nuclear Ah receptor. The more effective inhibition of induced EROD activity by I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and DIM are partial Ah receptor antagonists in the T47D human breast cancer cell line.

Breast Neoplasms↗

PubMed1996

Substituted flavones as aryl hydrocarbon (Ah) receptor agonists and antagonists.

The structure-dependent aryl hydrocarbon (Ah) receptor agonist and antagonist activities of the following substituted flavones were investigated: flavone, 4'-methoxy-, 4'-amino-, 4'-chloro-, 4'-bromo-, 4'-nitro-, 4'-chloro-3'-nitro-, 3'-amino-4'-hydroxy-, 3',4'-dichloro-, and 4'-iodoflavone. The halogenated flavones exhibited competitive Ah receptor binding affinities (IC50 = 0.79 to 2.28 nM) that were comparable to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (1.78 nM). The compounds also induced transformation of the rat cytosolic Ah receptor and induced CYP1A1 gene expression in MCF-7 human breast cancer cells. However, despite the high Ah receptor binding affinities for these responses, the halogenated flavones were > 1000 times less active than TCDD for the other responses. Moreover, for other substituted flavones, there was no correlation between Ah receptor binding affinities and their activities as Ah receptor agonists. For example, 4'-aminoflavone induced CYP1A1 mRNA levels in MCF-7 cells but exhibited relatively low Ah receptor binding affinity (IC50 = 362 nM) and did not induce transformation of the rat cytosolic Ah receptor. All of the substituted flavones inhibited TCDD-induced transformation of the Ah receptor, and 4'-iodoflavone, an Ah receptor agonist at high concentrations (1-50 microM), inhibited the transformation at concentrations as low as 0.05 and 0.5 microM. Subsequent interaction studies with TCDD and 4'-iodoflavone confirmed that the latter compound inhibits induction of CYP1A1 gene expression by TCDD in MCF-7 cells. The results obtained for the substituted flavones suggest that within this structural class of compounds, various substituent groups can affect markedly the activity of each individual congener as an Ah receptor agonist or antagonist. These substituent-dependent differences in activity may be related to ligand-induced conformational changes in the Ah receptor complex and/or support the proposed existence of more than one form of the Ah receptor.

Animals↗

PubMed1996

Isolation and characterization of variant benzo[a]pyrene-resistant T47D human breast-cancer cells.

T47D human breast cancer cells were grown in 1 microM benzo[a]pyrene (BaP) for 3.5 months, and 2 BaP-resistant (BaPr) variant cell lines (CS and C10) were isolated. Decreased aryl hydrocarbon (Ah)-responsiveness in the CS and C1O BaPr cells was characterized by lower (80 to 900/o) induction of CYP1A1-dependent activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), lower levels of the nuclear Ah receptor complex and significantly decreased Ah receptor mRNA levels. Nuclear estrogen receptor (ER) binding and ER mRNA levels were similar in wild-type and mutant cell lines, whereas epidermal growth factor receptor mRNA levels were significantly decreased in the variant BaPr T47D cells. 17beta-Estradiol induced proliferation of both wild-type and BaPr T47D cells, and TCDD inhibited this response but did not down-regulate nuclear ER levels. The unique characteristics of the BaPr T47D variant cells will be used to further elucidate the mechanism of interaction between the ER and Ah receptor signalling pathways.

Base Sequence↗

PubMed1996

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on insulin-induced responses in MCF-7 human breast cancer cells.

Insulin stimulated proliferation of MCF-7 human breast cancer cells in serum-free medium, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) did not affect cell growth. In cells cotreated with insulin plus TCDD or TCDF, insulin-induced cell proliferation and [3H]thymidine incorporation were inhibited. In contrast, alpha-naphthoflavone, a partial aryl hydrocarbon (Ah) receptor antagonist, blocked the inhibitory effects of TCDD, suggesting that the Ah receptor was involved in TCDD-induced responses in MCF-7 cells. TCDD alone did not affect Kd and Bmax values for binding of [125I]insulin to the insulin receptor (IR); however, in MCF-7 cells cotreated with insulin plus TCDD, the insulin-induced Kd value for IR-ligand binding was decreased and the Bmax value was increased. TCDD induced IR mRNA levels and inhibited several other insulin-induced responses including c-fos protooncogene expression, phosphorylation of the insulin receptor, and a 185-kDa protein in MCF-7 cells.

Base Sequence↗

PubMed1996

Effects of developmental exposure to indole-3-carbinol or 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring.

Treatment of pregnant female Sprague-Dawley rats on Gestational Day 15 with a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.5, 1.0, or 2.0 micrograms/kg) or indole-3-carbinol (I3C, 1.0 or 100 mg/kg), an aryl hydrocarbon (Ah) receptor agonist which is found in cruciferous vegetables, resulted in reproductive abnormalities in the male offspring (three to five litters in each treatment group). Anogenital distance and crown to rump length were altered by both compounds; however, the timing of the effects (Day 1 or 5) was variable and the responses were not necessarily dose-dependent. In 62-day-old offspring, seminal vesicle (24 to 26%), prostate (32 to 44%), testicular parenchymal (14%), and epididymal weight (19%) were decreased by one or more doses of TCDD. In contrast, I3C at one or more doses decreased daily sperm production/g testicular parenchyma (13 to 20%) and daily sperm production/testis (22%). Total number of sperum in the epididymis was significantly decreased (30 to 33%) in rats perinatally exposed to TCDD and this was due to a decreased (49 to 51%) number of sperm in the tail of the epididymis. Perinatal exposure to I3C did not affect any of these parameters. TCDD did not affect epididymal transit time of sperm through the complete epididymis at any of the doses (0.5 to 2.0 micrograms/kg). However, at the two highest doses (1.0 and 2.0 micrograms/kg), TCDD increased epididymal transit rate of sperm through the tail of the epididymis by 33 and 37%, respectively. In contrast, primarily due to decreased transit rate (39%) of sperm through the head plus body of the epididymis. I3C (1 mg/kg) significantly increased total epididymal transit time by 31%. In conclusion, perinatal exposure of pregnant rats to I3C, an Ah receptor agonist similar to TCDD, causes reproductive abnormalities in male rat offspring; however, I3C and TCDD elicited both common and different responses.

Animals↗

PubMed1996

Effect of transient expression of the oestrogen receptor on constitutive and inducible CYP1A1 in Hs578T human breast cancer cells.

Hs578T human breast cancer cells are an oestrogen receptor (ER)-negative cell line. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in formation of a 6.9 S nuclear aryl hydrocarbon (Ah) receptor complex, which bound to a [32P]dioxin-responsive element in a gel electrophoretic mobility shift assay. However, TCDD does not induce CYP1A1 gene expression or chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with pRNH11c or pMCAT5.12, which are Ah-responsive plasmids derived from the 5'-flanking region of the human and murine CYP1A1 genes respectively. Restoration of Ah responsiveness was investigated by co-transfecting Hs578T cells with pRNH11c or pMCAT5.12 and plasmids that express the ER (hER), Ah receptor (AhR) and AhR nuclear translocator (Arnt) proteins. ER expression resulted in significantly increased basal CAT activity; however, TCDD did not induce CAT activity in the transiently transfected cells. Expression of the AhR or Arnt proteins did not alter basal or inducible CAT activity. Expression of N- or C-terminal truncated ER in Hs578T resulted in differential regulation of Ah responsiveness. In Hs578T cells transiently expressing the ER, which contains C-terminal deletions (amino acids 282-595), basal CAT activity was also increased; however, Ah responsiveness was not restored. In contrast, transient expression of N-terminal-deleted (amino acids 1-178) ER resulted in a marked decrease in basal CAT activity but a restoration of Ah responsiveness. These results suggest that basal and inducible CAT activity in Hs578T cells transiently transfected with pRNH11c is modulated differentially by ER domains that are present in the N- and C-terminal regions of the ER.

Base Sequence↗

PubMed1996

Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression.

Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-[32P]Sp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-[32P]Sp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.

Antibodies↗

PubMed1996

Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro.

The potential estrogenic activities of atrazine and simazine were investigated in vivo using the immature female Sprague-Dawley rat uterus and in vitro using the estrogen-responsive MCF-7 human breast cancer cell line and the estrogen-dependent recombinant yeast strain PL3. Animals that were dosed with 50, 150, or 300 mg/kg of atrazine or simazine alone for 3 consecutive days did not exhibit any significant increases in uterine wet weight while decreases in cytosolic progesterone receptor (PR) binding levels and uterine peroxidase activity were observed. 17 beta-estradiol (E2)-induced increases in uterine wet weight were not significantly affected by cotreatment with either chemical; however, some dose-independent decreases in E2-induced cytosolic PR binding and uterine peroxidase activity were observed. In vitro, atrazine and simazine did not affect basal or E2-induced MCF-7 cell proliferation or the formation of nuclear PR-DNA complexes as determined by gel electrophoretic mobility shift assays. In addition, these chloro-S-triazines did not display agonist activity or antagonize E2-induced luciferase activity in MCF-7 cells transiently transfected with a Gal4-human estrogen receptor chimera (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc). Moreover, the estrogen-dependent PL3 yeast strain was not capable of growth on minimal media supplemented with atrazine or simazine in place of E2. Collectively, these results indicate that the reported estrogenic and antiestrogenic effects elicited by these chemicals are not mediated by the estrogen receptor.

Animals↗

PubMed1995

Structure-dependent induction of CYP2B by polychlorinated biphenyl congeners in female Sprague-Dawley rats.

The dose-response induction of hepatic microsomal pentoxyresorufin O-dealkylase (PROD) activity by phenobarbital (PB) and several polychlorinated biphenyl (PCB) mixtures and congeners was determined in the immature female Sprague-Dawley rat. At a dose of 75 mg/kg/day of PB for 3 days, the microsomal PROD activity was 2154 pmol/min/mg protein. Aroclors 1260, 1254, 1242, and 1016 did not induce maximal PROD activity at doses up to 500 mg/kg, and only Aroclor 1016 induced > a half-maximal response at the 500 mg/kg dose. The relative potencies of eighteen different PCB congeners were also determined, and the structures of these compounds differed with respect to the degree of chlorination (tri- to octochloro) and substitution patterns. The relative potencies of these compounds were estimated by comparing their induced activities at the high dose (150 or 100 mg/kg) with that of PB. The most potent inducers were 2,3,3',4',5,6-hexaCB and 2,2',3,4',5,5',6-heptaCB; at a dose of 150 mg/kg, the PROD activity induced by 2,2'3,4',5,5',6-heptaCB was comparable to that observed for PB. 2,3,3',4',5,6-HexaCB was the most potent inducer, and hepatic PROD activity in rats treated with 150 mg/kg was 4202 pmol/min/mg; this value was higher than that observed for PB at a dose of 75 mg/kg. A second group of congeners including 2,2',3,4,4',5,5'-heptaCB, 2,2',4,4',5,5'-hexaCB, 2,2',3,3',4,4',5,5'-octaCB 2,2',4,4'-tetraCB, 2,2',4,5,5'-pentaCB, 2,2',3,4,4',5',6-heptaCB, 2,2',4,4',5-pentaCB and 2,2',3,3',4',5,5',6-octaCB induced PROD activity > or = 1090 pmol/min/mg at the 150 mg/kg dose, and this value was > 50% of the maximal response observed for PB. The remaining compounds, namely 2,4,4'-triCB, 2,2',3,4'-tetraCB, 2,2',5,5'-tetraCB, 2,3',4,4',5-pentaCB, 2,3,3',4,4'-pentaCB, 2,2',4,4',5,6'-hexaCB, 2,3,3',4,4',5,5'-heptaCB and 2,2',3,3',4,4,5-heptaCB were all relatively weak inducers of hepatic microsomal PROD activity ( < 450 pmol/min/mg). In parallel experiments, western blot analysis of immunoreactive CYP2B1 and CYP2B2 protein showed that PB, the PCB mixtures, and congeners induced both proteins. Previous studies have identified a cis-acting DNA element that plays a role in regulating CYP2B1/B2 gene expression and binds nuclear trans-acting factor(s) induced by PB. The results of gel electrophoretic mobility shift assays with nuclear extracts showed that both PB and 2,2',3,4',5,5',6-heptaCB induce formation of a common retarded band using a 32P-labeled oligonucleotide corresponding the the cis-acting DNA promoter sequence. Both PB and PCBs appear to induce CYP2B1/B2 via a common mechanism. Although the results of this study do not define structure-induction (CYP2B1/B2) relationships for PCBs, two compounds, namely 2,3,3',4',5,6-hexaCB and 2,2',3,4',5,5',6-heptaCB, were identified as highly potent inducers.

Animals↗

PubMed1995

Naringenin: a weakly estrogenic bioflavonoid that exhibits antiestrogenic activity.

Treatment of immature 21-day-old female Sprague-Dawley rats with 17 beta-estradiol (E2) (0.5 microgram/rat) caused a significant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mg/rat, the bioflavonoid naringenin did not cause a significant increase in any of these E2-induced responses. However, in rats cotreated with E2 (0.5 microgram/rat) plus naringenin (30 mg/rat); there was a significant decrease in E2-induced uterine wet weight, DNA synthesis, PR binding, and peroxidase activity, indicating that naringenin exhibits antiestrogenic activity in the immature rodent uterus. The binding of uterine nuclear extracts to a 32P-labeled estrogen responsive element (ERE) or progesterone responsive element (PRE) was determined using gel electrophoretic band shift assays. Incubation of [32P]ERE with uterine nuclear extracts from rats treated with naringenin or E2 resulted in the formation of estrogen receptor (ER):ERE complexes; a higher mobility complex was prominent in the extracts from E2-treated rats, whereas a lower mobility complex was observed using nuclear extracts from naringenin-treated animals. There was a significant decrease in the intensity of the E2-induced complex using nuclear extracts from rats treated with E2 plus naringenin. In contrast, transformed cytosol from control rats gave an intense ER:ERE complex, whereas the intensity of the band was decreased markedly using transformed uterine cytosol from treated rats. Formation of a PR:PRE complex was also determined using transformed uterine cytosol. Cytosol from E2-treated rats gave an intense retarded band, whereas only weak bands were observed using cytosols from DMSO- (solvent), naringenin-, or naringenin plus E2-treated cells. The results of in vitro studies showed that 1 nM E2 increased (3- to 4-fold) the growth of MCF-7 human breast cancer cells, whereas 1-1000 nM naringenin had no effect on cell proliferation. In cells cotreated with 1 nM E2 plus 1000 nM naringenin, there was a significant decrease in E2-induced cell growth. In MCF-7 cells transiently transfected with a pS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 microM naringenin plus 1 nM E2, naringenin inhibited E2-induced luciferase activity. The results of these studies confirmed that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells.

Animals↗

PubMed1995

Comparative properties of the nuclear aryl hydrocarbon (Ah) receptor complex from several human cell lines.

The aryl hydrocarbon (Ah) responsiveness of the T-47D, Hep G2, LS180, MCF-7, A431, C-4II and MDA-MB-231 human cancer cell lines was determined by the induction of CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity. With the exception of teh MDA-MB-231 breast cancer cell line, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly induced CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity in the remaining six cell lines and, based on their EC50 values, for ethoxyresorufin O-deethylase induction, their Ah responsiveness followed the order T-47D > C-4II > MCF-7 > LS180 > HEP G2 > A431. In contrast, all the cell lines expressed the nuclear Ah receptor complex (167.1-24.5 fmol/mg protein) which bound to a 32P-labeled consensus dioxin responsive element (DRE) in a gel mobility shift assay. The results of gel permeation chromatography a sucrose density gradient centrifugation studies showed that the calculated Mr values for the nuclear Ah receptor complex varied from 175 kDa (MDA-MB-231 cells) to 221 kDa and the apparent molecular weight of the nuclear Ah receptor complex cross-linked to a bromodeoxyuridine-substituted DRE was 200 kDa. The data show that the molecular properties and levels of the nuclear Ah receptor complex from seven different human cancer cell lines do not predict Ah responsiveness.

Affinity Labels↗

PubMed1995

In vitro inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced activity by alpha-naphthoflavone and 6-methyl-1,3,8-trichlorodibenzofuran using an aryl hydrocarbon (Ah)-responsive construct.

Rat hepatoma H4IIE and mouse hepatoma Hepa 1c1c7 cells were transiently transfected with a plasmid construct that contained the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the mouse mammary tumor virus promoter and one copy of the dioxin responsive element. Treatment of transfected H4IIE and Hepa 1c1c7 cells with 10(-13) to 10(-6) M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a concentration-dependent increase in transient CAT activity. Maximum CAT activity was induced in both cell lines by exposure to 10(-9) M TCDD. The induction of CAT activity correlated well with the TCDD-induced, P4501A1-dependent ethoxyresorufin O-deethylase activity. Cotreatment of transfected cells with 10(-9) M TCDD and 10(-8) to 10(-6) M alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) resulted in a concentration-dependent reduction of TCDD-induced CAT activity. Treatment of cells with 10(-6) M alpha NF or MCDF alone resulted in only minimal induction of CAT activity. Both antagonists inhibited the induction of genes under the control of the CYP1A1 and mouse mammary tumor virus promoters, which indicates that the alpha NF- and MCDF-mediated antagonism of TCDD-induced, aryl hydrocarbon receptor-dependent gene transcription does not depend on promoter context.

Animals↗

PubMed1995

Induction of Cyp1a-1 and Cyp1a-2 gene expression by a reconstituted mixture of polynuclear aromatic hydrocarbons in B6C3F1 mice.

The potential non-additive interactions of polynuclear aromatic hydrocarbon (PAH) mixtures as inducers of Cyp1a-1 and Cyp1a-2 gene expression were investigated in B6C3F1 mice using a reconstituted PAH mixture. The chemical composition (% by weight) of the reconstituted PAH mixture was: 2-ring PAHs--indan (0.22), naphthalene (23.8), 2-methylnaphthalene (23.2) and 1-methylnaphthalene (13.3); 3-ring PAHs--acenaphthylene (7.7), acenaphthene (0.6), dibenzofuran (0.7), fluorene (4.3), phenanthrene (10.5) and anthracene (3.4); > or = 4-ring PAHs--fluoranthene (2.4), pyrene (4.3), benz[a]anthracene (1.4), chrysene (1.5), benzo[b]fluoranthene (0.8), benzo[k]fluoranthene (0.9) and benzo[a]pyrene (0.9). The composition of the 2-, 3- and > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fraction and reconstituted mixture induced hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity and Cyp1a-1 mRNA levels, whereas the 2- and 3-ring PAHs were only weakly active. Direct comparison of the potencies of the reconstituted mixture and > or = 4-ring PAHs showed that the Cyp1a-1 induction activity of the reconstituted mixture was due to the > or = 4-ring PAHs. The reconstituted PAH mixture and > or = 4-ring PAHs also induced Cyp1a-2 hepatic mRNA levels and microsomal methoxyresorufin O-deethylase (MROD) activity; however, their dose-response curves indicated that the reconstituted PAH mixture was more potent as a Cyp1a-2 inducer than the > or = 4 ring PAHs. The differences in potency were due to 3-ring PAHs which were found to be strong inducers of hepatic Cyp1a-2 mRNA levels and microsomal MROD activity at the lowest dose administered (37 mg/kg). The 3-ring mixture caused a maximal 29-fold increase in hepatic MROD activity at a dose of 292 mg/kg, but only 28% of maximal induction of EROD activity. Northern analysis of liver mRNA from mice treated with 3-ring PAHs showed that there was minimal induction of Cyp1a-1 mRNA levels. The 3-ring PAHs did not competitively bind to the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor suggesting that 3-ring PAHs are a new class of Cyp1a-2 inducers which do not act through the Ah receptor.

Animals↗

PubMed1995

Halogenated aromatic hydrocarbon-induced suppression of the plaque-forming cell response in B6C3F1 splenocytes cultured with allogenic mouse serum: Ah receptor structure activity relationships.

The immunosuppressive effects of halogenated aromatic hydrocarbons (HAHs) were investigated in B6C3F1 female mice and in mouse splenocytes cultured with allogenic mouse serum using the Mishell-Dutton model for in vitro immunization to trinitrophenyl-lipopolysaccharide (TNP-LPS). Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 1,2,3,7,8-PeCDF, 1,3,6,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (pentaCB), or 3,3',4,4',5,5'-hexaCB resulted in a dose-dependent suppression of the splenic plaque-forming cell (PFC) response both in vivo and in vitro. The effective dose required to decrease 50% (ED50) of the response to 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDF, 1,3,6,8-TCDF, 3,3',4,4',5-pentaCB, or 3,3',4,4',5,5'-hexaCB in vivo was 14.1, 5.5, 1695, 34,800, 21, and 19 nmol/kg, respectively, and in vitro was 7.0, 10.6, 149, 2325, 9.1 and 9.1 nM, respectively. There was an excellent rank order and linear correlation between the in vivo versus in vitro activities for these HAHs (r < 0.99) and the relative immunosuppressive potencies of these compounds paralleled their binding affinities for the aryl hydrocarbon (Ah) receptor. These results show that splenocytes cultured with allogenic mouse serum is an Ah-responsive in vitro assay which can be used for quantitating the immunosuppressive effects of HAHs.

Analysis of Variance↗

PubMed1995

Identification of 3'-methoxy-4'-nitroflavone as a pure aryl hydrocarbon (Ah) receptor antagonist and evidence for more than one form of the nuclear Ah receptor in MCF-7 human breast cancer cells.

The competitive binding of 3'-methoxy-4'-nitro, 4'-amino-3'-methoxy, 4'-methoxy-3'-nitro, and 3'-amino-4'-methoxyflavone (compounds 1 to 4, respectively) to the rat cytosolic aryl hydrocarbon (Ah) receptor gave IC50 values of 2.27, 86.1, 872, and 19.4 nM. Flavones 3 and 4 were characterized as Ah receptor agonists in MCF-7 human breast cancer cells and induced CYP1A1 gene expression, whereas the 3-methoxy-substituted flavones (1 and 2) were inactive. All four compounds inhibited induction of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells; moreover, in vitro studies with TCDD-induced rat liver microsomes showed that flavones 1 to 4 inhibited EROD activity in the presence or absence of NADPH. In MCF-7 cells cotreated with flavones 1 or 2 (0.01 to 10 microM) plus TCDD or [3H]TCDD, there was a concentration-dependent inhibition of TCDD-induced CYP1A1 mRNA levels and formation of radiolabeled nuclear Ah receptor complex. Velocity sedimentation analysis of nuclear extracts from MCF-7 cells treated with [3H]TCDD plus 1 or 10 microM concentrations of flavones 1 and 2 showed that an early eluting specifically bound nuclear Ah receptor complex was present. However, under these same conditions the flavones inhibited TCDD-induced CYP1A1 gene expression. The apparent molecular mass of this nuclear complex was 190 kDa as determined by cross-linking to a 32P-labeled bromodeoxyuridine-substituted consensus dioxin-responsive element. Similar cross-linking results were obtained using the nuclear extract from MCF-7 cells treated with [3H]TCDD alone. The results of this study suggest that there are at least two forms of the nuclear Ah receptor complex in MCF-7 cells; the major transcriptionally active form binds [3H]TCDD and flavones 1 or 2 inhibit nuclear uptake of this receptor complex. The other form of the nuclear Ah receptor complex appears to be transcriptionally inactive and ligand binding with [3H]TCDD is not competitively inhibited by flavones 1 and 2.

Animals↗

PubMed1995

Immunosuppressive activity of polychlorinated biphenyl mixtures and congeners: nonadditive (antagonistic) interactions.

The dose-response inhibition of the splenic plaque-forming cell (PFC) response and serum IgM units to the antigen, trinitrophenyl-lipopolysaccharide, was determined for several polychlorinated biphenyl (PCB) mixtures and congeners in female B3C3F1 mice. The ED50 values for Aroclor 1260-, 1254-, 1248-, and 1242-induced immunotoxicity varied by less than twofold from 355 to 699 mg/kg. The range of ED50 values for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5-pentaCB, 3,3',4,4',5,5'-hexaCB, 2,3,3',4,4'-pentaCB, 2,3',4,4',5-pentaCB, 2,3,3',4,4',5-hexaCB, 2,3,3',4,4',5,5'-heptaCB, 2,2',3,3',4,4',5-heptaCB, and 2,2',3,4,4',5,5'-heptaCB were 4.6 to 4.9, 134 to 245, 4.7 to 7.0, 6.9 to 11.1, 88,000 to 121,000, 122,000 to 132,000, 99,000 to 157,000, 89,000 to 129,000, 117,000 to 240,000, and 132,000 to 238,000 micrograms/kg, respectively. The immunotoxicity-derived toxic equivalency factors (TEFs) for these congeners could be calculated from the ED50 (TCDD)/ED50 (congener) ratios and the TEF values were within the range of those previously determined for other aryl hydrocarbon receptor-mediated responses. Based on the known concentrations of these congeners in the PCB mixtures, TCDD or toxic equivalents (TEQs) in the mixture were calculated [i.e., TEQ = sigma (PCBcongener x TEF)] using the immunotoxicity-derived TEFs (plaque-forming cells/10(6) viable cells). TEQ values for Aroclors 1260, 1254, 1248, and 1242 were 16.0, 54.4, 260.4, and 197 ppm, respectively. Based on the ED50 value for the immunosuppressive activity of TCDD (4.8 micrograms/kg), the calculated ED50 values for immune suppression by Aroclors 1260, 1254, 1248, and 1242 were 300, 88, 18, and 24 mg/kg, respectively. The ED50 (observed)/ED50 (calculated) ratios were 1.2, 5.9, 21, and 22.0 for Aroclors 1260, 1254, 1248 and 1242, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Alkyl polychlorinated dibenzofurans and related compounds as antiestrogens in the female rat uterus: structure-activity studies.

The antiestrogenic activity of a series of alkyl-substituted polychlorinated dibenzofurans (PCDFs) were determined in the immature female Sprague-Dawley rat uterus. The compounds utilized in this study contain two, three, or four lateral substitutents and include: 6-methyl-1,3,8-triCDF, 6-ethyl-1,3,8-triCDF, 6-n-propyl-1,3,8-triCDF, 6-i-propyl-1,3,8-triCDF, 6-t-butyl-1,3,8-triCDF, 8-methyl-1,3,6-triCDF (two lateral substituents); 6-methyl-2,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, 8-methyl-1,3,7-triCDF, and 8-methyl-1,2,4,7-tetraCDF (three lateral substituents); 8-methyl-2,3,7-triCDF, 8-methyl-2,3,4,7-tetraCDF (four lateral substituents). Two additional compounds, 8-methyl-2,3,7-trichlorodibenzo-p-dioxin and 8-methyl-2,3,7-tribromodibenzo-p- dioxin (four lateral substituents), were also utilized. All of the alkyl-substituted compounds inhibited estrogen-induced uterine wet weight increase and cytosolic and nuclear progesterone and estrogen receptor binding. The effects of structure on the antiestrogenic potencies were determined using 6-i-propyl-1,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, and 8-methyl-2,3,4,7-tetraCDF as representative congeners containing two, three, and four lateral substituents, respectively. The ED50 values for antiestrogenicity were similar for the three compounds; however, the ED50 values for induction of hepatic CYP1A1-dependent activity were 73,600 (estimated), 8.52, and 5.31 mumol/kg for 6-i-propyl-1,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, and 8-methyl-2,3,4,7-tetraCDF, respectively. Since CYP1A1 can be used as a surrogate for toxic potency in the rat then high ED50 (induction)/ED50 (antiestrogenicity) ratios would be indicative of low toxicity and high antiestrogenic potency. The ratio was 13,990 to 17,100 for 6-i-propyl-1,3,8-triCDF, whereas the corresponding value for the compounds with three or four lateral substituents varied from 0.64 to 3.34. The results suggests that the 1,3,6,8-substituted alkyl PCDFs are useful structural models for developing new aryl hydrocarbon receptor-mediated antiestrogens for future clinical use as antiestrogens.

Animals↗

PubMed1995

Cellular and molecular biology of aryl hydrocarbon (Ah) receptor-mediated gene expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds elicit diverse toxic and biochemical responses in laboratory animals and mammalian cells in culture. TCDD induces CYP1A1 gene expression and results of extensive research have delineated the molecular mechanism of this response. In target cells, TCDD initially binds to the aryl hydrocarbon (Ah) receptor which accumulates in the nucleus as an Ah-receptor:aryl hydrocarbon nuclear translocator (Arnt) protein heterodimeric complex. The nuclear Ah receptor complex acts as a ligand-induced transcription factor which binds to transacting genomic dioxin/xenobiotic responsive elements (DREs/XREs) located in the 5'-regulatory region upstream from the initiation start site and this interaction results in transactivation of gene transcription. DREs have been identified in several other genes which are induced by TCDD, including CYP1A2, aldehyde-3-dehydrogenase, NAD(P)H quinone oxidoreductase, and glutathione S transferase Ya and similar induction response pathways have been observed or proposed. However, TCDD and other Ah receptor agonists also inhibit expression of several genes and research in this laboratory has investigated inhibition of estrogen (E2)-induced genes including uterine epidermal growth factor, c-fos protooncogene, and the progesterone receptor, estrogen receptor (ER) and cathepsin D genes in human breast cancer cell lines. In MCF-7 human breast cancer cells, E2 induces cathepsin D gene expression and this is associated with formation of an ER/Sp1 complex at the sequence in the promoter region (-199/-165) of this gene. Within 30 min TCDD causes a rapid inhibition of E2-induced cathepsin D gene expression in MCF-7 cells. Moreover, using a series of synthetic oligonucleotides which include the wild-type ER/Sp1 and various mutants, it was shown by gel electromobility shift and transient transfection assays that the nuclear Ah receptor complex binds to an imperfect DRE located between the ER and Sp1 binding sequences. This interaction results in disruption of the ER/Sp1 complex and inhibition of E2-induced gene expression. These results illustrate that the nuclear Ah receptor complex also exhibits activity as a negative transcription factor via a mechanism which is similar to that reported for Ah receptor-mediated induction of gene expression.

Animals↗