Methyl-substituted diindolylmethanes as inhibitors of estrogen-induced growth of T47D cells and mammary tumors in rats.

Diindolylmethane (DIM) is formed by acid catalyzed dimerization of the phytochemical indole-3-carbinol, and both compounds inhibit formation and/or growth of mammary tumors in rodents. In this study, we have investigated the aryl hydrocarbon receptor (AhR) agonist activity and inhibitory AhR-estrogen receptor crosstalk induced by the following methyl-substituted DIMs: 1,1'-dimethyl-, 2,2'-dimethyl-, 5,5'-dimethyl-, 6,6'-dimethyl-, and 7,7'-dimethylDIM and 1,1',2,2'-tetramethylDIM. The six compounds bound to the rat cytosolic AhR in a transformation assay but, at concentrations < or = 10 microM, exhibited minimal to non-detectable AhR agonist or antagonist activities associated with CYP1A1 induction. In contrast, the methyl-substituted DIMs inhibited estrogen-induced T47D human breast cancer cell growth and the four most active compounds (1,1'-, 2,2'-, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM) inhibited one or more estrogen-induced responses in the 21-day-old female B6C3F1 mice at a dose of 100 mg/kg/day (X3). Induction of hepatic CYP1A1-dependent activity was not observed at this high dose. The antitumorigenic activity of these compounds was examined in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor model in which the DIM analogs were orally administered (by gavage in corn oil) at a dose of 1 mg/kg/day (X10). 1,1'-DimethylDIM, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM significantly inhibited mammary tumor growth, and this was not accompanied by changes in organ/body weights or histopathology. These studies demonstrate that methyl-substituted DIMs are selective AhR modulators (SAhRMs) with potential for clinical treatment of breast cancer.

Tamoxifen-induced antitumorigenic/antiestrogenic action synergized by a selective aryl hydrocarbon receptor modulator.

Tamoxifen (TAM) is a highly effective selective estrogen receptor (ER) modulator used extensively for the treatment and prevention of breast cancer. However, prolonged treatment of women with TAM may be a risk factor for endometrial cancer, and research in our laboratory is focused on the development of selective aryl hydrocarbon receptor modulators that can be used in combination with TAM to improve its efficacy in the breast and inhibit TAM-induced endometrial effects. This study investigated the effects of the selective aryl hydrocarbon receptor modulators 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF) alone and in combination with TAM in the carcinogen-induced mammary tumor model and in the ovariectomized uterotropic assay using female Sprague Dawley rats. The lowest effective dose of 6-MCDF that inhibited tumor growth was 50 microg/kg/day, and TAM was antitumorigenic at a dose of 100 microg/kg/day. In animals cotreated with TAM + 6-MCDF at doses of 100, 50, or 25 microg/kg/day of each compound, complete inhibition of mammary tumor growth was observed at all doses, and the results are consistent with a more than additive antitumorigenic response for the low dose group (25 + 25 microg/kg) and additive interactions at the 50 and 100 microg/kg doses. In a separate experiment, 6-MCDF (800 microg/kg) inhibited TAM-induced peroxidase activity and progesterone receptor binding in the ovariectomized rat uterus but did not affect TAM-induced bone growth in ovariectomized rats. This study also investigated the effects of TAM and 6-MCDF alone and in combination on ERalpha protein levels in MCF-7 human breast cancer cells as a model for studying interactions between these compounds. The results show that 6-MCDF decreased TAM-induced ERalpha levels in the absence or presence of 17beta-estradiol through proteasome activation, and these interactions may contribute to the observed combined antitumorigenic effects of these compounds.

Inhibition of carcinogen-induced rat mammary tumor growth and other estrogen-dependent responses by symmetrical dihalo-substituted analogs of diindolylmethane.

90%) by the haloDIMs at concentrations of 5 or 10 microM, and only 4, 4'-dichloroDIM alone increased cell proliferation. With the exception of 5,5'-difluoroDIM, the remaining compounds also inhibited E2-induced growth of MCF-7 human breast cancer cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic in the immature female B6C3F1 mouse uterus; however, in animals co-treated with E2 (0.02 microg/mouse), 5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine progesterone receptor (PR) binding and uterine peroxidase activity, whereas 5,5'-dichloro- and 5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter response. The antitumorigenic activities of the dihaloDIMs were determined by their inhibition of carcinogen-induced mammary tumor growth in female Sprague-Dawley rats. 4,4'-Dichloro-, 5,5'-dibromo- and 6,6'-dichloroDIM, significantly inhibited mammary tumor growth at doses of 1 mg/kg every second day, and no significant changes in organ weights or liver and kidney histopathology were observed. These three compounds were more active than DIM in the same in vivo assay.

3,3'4,4'-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells.

3,3',4,4'-Tetrachlorobiphenyl (tetraCB) binds to the aryl hydrocarbon receptor (AhR), and several reports have demonstrated that AhR agonists exhibit antiestrogenic and antitumorigenic activities in human breast cancer cells, the rodent uterus and breast. In contrast, a recent study showed that 3,3',4,4'-tetraCB bound the estrogen receptor (ER) and exhibited ER agonist activities, and we therefore have reinvestigated the estrogenic and antiestrogenic activities of 3,3',4,4'-tetraCB. Our results showed that 3,3',4,4'tetraCB and a structurally related analog, 3,3',4,4',5-pentaCB, did not bind the mouse uterine or human ER, did not induce proliferation of MCF-7 or T47D human breast cancer cells or induce reporter gene activity in cells transfected with E2-responsive constructs derived from the creatine kinase B (pCKB) or cathepsin D (pCD) gene promoters. Moreover, 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB did not induce an increase in uterine wet weight, peroxidase activity or progesterone receptor binding in the 21-25-day-old female B6C3F1 mouse uterus. In contrast, both compounds inhibited 17beta-estradiol (E2)-induced cell proliferation and transactivation in MCF-7/T47D cells and uterine responses in B6C3F1 mice; surprisingly inhibition of E2-induced reporter gene activity was not observed in T47D cells transfected with pCKB, and this was observed as a cell-specific response with other AhR agonists. Additionally, 3,3',4,4'-tetraCB significantly inhibited mammary tumor growth in female Sprague-Dawley rats initiated with 7,12-dimethylbenzanthracene. Our results indicate that 3,3',4,4'-tetraCB does not exhibit ER agonist activity but exhibits a broad spectrum of antiestrogenic responses consistent with ligand-mediated AhR-ER crosstalk.

Development of selective aryl hydrocarbon receptor modulators for treatment of breast cancer.

The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix DNA-binding protein that forms a transcriptionally-active heterodimer with the AhR nuclear translocator (Arnt) protein. The nuclear AhR complex is a ligand-induced transcription factor and the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a high affinity ligand for the AhR. TCDD induces a diverse spectrum of tissue-, sex- and species-specific biochemical and toxic responses in Ah-responsive cells/tissues including the inhibition of 17beta-oestradiol (E2)-induced gene expression in the rodent uterus and mammary and in human breast cancer cell lines. TCDD also inhibits spontaneous and carcinogen-induced mammary tumour formation and growth in rodent models. Research in this laboratory has utilised the AhR as a target for developing anticancer drugs for treatment of breast cancer and two different structural classes of selective AhR modulators (SAhRMs) have been developed. Alternate-substituted (1,3,6,8- and 2,4,6,8-) alkyl polychlorinated dibenzofurans (PCDFs) and substituted diindolylmethanes (DIMs) bind the AhR and induce a pattern of AhR-oestrogen receptor (ER) inhibitory cross-talk similar to that observed for TCDD including inhibition of mammary tumour growth at doses < 1.0 mg/kg/day. In contrast, effective doses of these compounds do not induce hepatic CYP1A1-dependent activity or other AhR-mediated toxic responses induced by TCDD. These results indicate that SAhRMs may be an important new class of drugs for clinical treatment of breast cancer via AhR-ER inhibitory cross-talk.

Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane.

Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of cruciferous vegetables which exhibit antitumorigenic activity associated with altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with 10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and induction of CYP1A1 gene expression was observed at concentrations >50 microM. Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats. Results of this study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells, reporter gene activity in cells transiently transfected with an E2-responsive plasmid (containing a frog vitellogenin A2 gene promoter insert) and down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and this was not accompanied by induction of hepatic CYP1A1-dependent activity. Thus, DIM represents a new class of relatively non-toxic AhR-based antiestrogens that inhibit E2-dependent tumor growth in rodents and current studies are focused on development of analogs for clinical treatment of breast cancer.

Induction of 16alpha-/2-hydroxyestrone metabolite ratios in MCF-7 cells by pesticides, carcinogens, and antiestrogens does not predict mammary carcinogens.

The effects of several pesticides, mammary carcinogens, and antiestrogens on 17beta-estradiol (E2), 16alpha- and 2-hydroxylase activities, and 16alpha-/2-hydroxyestrone (OHE1) ratios were investigated in MCF-7 cells using a radiometric assay. The mammary carcinogens 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (BaP), respectively, increased and decreased 16alpha-/2-OHE1 ratios at some concentrations. The 16alpha-/2-OHE1 metabolite ratios for 10(-5) M kepone, atrazine, p,p'-DDE, o,p'-DDE, o,p'-DDT, and beta-hexachlorocyclohexane were 1.82 +/- 0.060, 0.71 +/- 0.027, 0.66 +/- 0.030, 1.56 +/- 0.089, 1.14 +/- 0.059, and 0.69 +/- 0.052 (mean +/- standard error), respectively, and did not show any specific trend. The effects of a series of direct and indirect acting antiestrogens on 16alpha-/2-OHE1 metabolite ratios were also investigated, and the results were compound specific. Indole-3-carbinol, tamoxifen, 4'-hydroxytamoxifen, and 9-cis,retinoic acid decreased the ratio; the effects of all trans-retinoic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin were concentration dependent; the antiestrogen ICI 182,780 increased the 16alpha-/2-OHE1 metabolite ratio. The results indicate that in MCF-7 cells treated with pesticides, mammary carcinogens, and antiestrogens, there were both increased and decreased 16alpha-/2-OHE1 metabolite ratios for each class of chemicals and the assay did not predict mammary carcinogens.

Estrogenic and antiestrogenic activities of 16alpha- and 2-hydroxy metabolites of 17beta-estradiol in MCF-7 and T47D human breast cancer cells.

The comparative mitogenic activities of 17beta-estradiol (E2) and four metabolites, 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 16alpha-hydroxyestradiol (16alpha-OHE2) and 16alpha-hydroxyestrone (16alpha-OHE1) were determined in estrogen receptor (ER)-positive MCF-7 and T47D human breast cancer cells. E2 (1 nM) induced a 7- to 13-fold increase in cell number in both cell lines compared to untreated cells and the mitogenic potencies of 16alpha-OHE1 or 16alpha-OHE2 were comparable to or greater than E2. In contrast, 2-OHE1 and 2-OHE2 were weak mitogens in both cell lines and in cells cotreated with 1 nM E2 and 100 or 1000 nM 2-OHE1 or 2-OHE2, there was a significant inhibition of hormone-induced cell proliferation. The comparative ER agonist/antagonist activities of E2 and the metabolites on transactivation were determined in T47D cells transiently transfected with constructs containing promoter inserts from the cathepsin D (pCD) and creatine kinase B (pCKB) genes. E2, 16alpha-OHE2 and 16alpha-OHE1 induced reporter gene activity in both MCF-7 or T47D cells transfected with pCKB or pCD. In contrast, 2-OHE1 and 2-OHE2 did not exhibit ER agonist activity for these transactivation assays, but in cells cotreated with E2 plus 2-OHE1 or 2-OHE2, there was a significant decrease in the hormone-induced response. These results demonstrate that 16alpha-OHE1/16alpha-OHE2 exhibit estrogenic activities similar to that observed for E2, whereas the 2-catecholestrogens are weak ER agonists (cell proliferation) or antagonists (cell proliferation and transactivation).

Ah receptor agonists as endocrine disruptors: antiestrogenic activity and mechanisms.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds induce a broad spectrum of biochemical and toxic responses and disrupt multiple endocrine pathways. Research in this laboratory has focused on characterizing aryl hydrocarbon receptor (AhR)-mediated antiestrogenicity in the rodent uterus and mammary and in human breast cancer cells. TCDD inhibits multiple estrogen (E2)-induced responses in these tissues including development or growth of human mammary and endometrial cancer cells, carcinogen-induced mammary cancer in rats, and mammary cancer in mice bearing breast cancer cell xenografts. The mechanisms of AhR-mediated antiestrogenicity are complex; however, studies on the molecular biology of cross-talk between the AhR and estrogen-receptor (ER) signaling pathways have been initiated using several E2-regulated genes as models. The results indicate that the nuclear AhR complex targets specific genomic core inhibitory dioxin responsive elements (iDREs) in promoter regions of some E2-responsive target genes to inhibit hormone-induced transactivation. The pS2, cathepsin and c-fos genes have functional iDREs, whereas the iDRE in the progesterone receptor gene promoter was not functional. Research has also focused on development of AhR-based antiestrogens which inhibit mammary tumor development and growth but do not exhibit prototypical AhR-induced toxic responses.

Induction of estradiol 2-hydroxylase activity in MCF-7 human breast cancer cells by pesticides and carcinogens.

The induction of 17ß-estradiol (E2) 2-hydroxylase activity was investigated in MCF-7 human breast cancer cells using 2-[(3)H]E2 as the substrate in a radiometric assay. Treatment of MCF-7 cells with 10 µM indole-3-carbinol (I3C) for 48 h caused a 3.5-fold induction of E2 2-hydroxylase activity, whereas, I3C at concentrations as high as 100 µM did not induce CYP1A1 mRNA levels or immunoreactive protein. Thus, the induction of E2 2-hydroxylase activity using the radiometric assay was not dependent on induction of CYP1A1. E2 2-hydroxylase activity was also increased by I3C within 2 h after treatment suggesting in situ interactions with the cellular cytochrome P450 system. The time-dependent effects of various chlorinated pesticides, antiestrogens and mammary carcinogens on E2 2-hydroxylase activity were also investigated. p,p'-DDE, atrazine and the mammary carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) significantly decreased E2 2-hydroxylase activity after 2 h; whereas, only the latter two compounds decreased activity after 48 h. Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the mammary carcinogen benzo[a]pyrene (BaP) induced E2 2-hydroxylase in MCF-7 cells after incubation for 48 h and this was also paralleled by induction of CYP1A1 protein. The antiestrogens ICI 164 384 and ICI 182 780 decreased E2 2-hydroxylase activity in MCF-7 cells after incubation for 48 h, whereas tamoxifen and 4-hydroxytamoxifen were inactive. The results indicate that chemical-induced modulation of E2 2-hydroxylase activity in MCF-7 cells is complex and does not predict their activity as mammary carcinogens.

Inhibition of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor growth by aryl hydrocarbon receptor agonists.

The antitumorigenic activities of 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), 8-methyl-1,3,6-trichlorodibenzofuran (8-MCDF) and 6-cyclohexyl-1,3,8-trichlorodibenzofuran (6-CHDF) were investigated in the 7,12-dimethylbenz[a]anthracene (DMBA) rat mammary tumor model. At doses of 5, 10 or 25 mg/kg/week, both 6-MCDF and 8-MCDF significantly inhibited mammary tumor growth and at the 5 mg/kg/week dose >50% growth inhibition was observed. In contrast, 6-CHDF was inactive at the 5 mg/kg/week dose and the structure-antitumorigenicity relationships (6-MCDF/8-MCDF > 6-CHDF) correlated with structure-antiestrogenicity (rat uterus) studies and the relative binding affinities of these compounds for the aryl hydrocarbon receptor (AhR). The antitumorigenic activity of 6-MCDF or 8-MCDF in the mammary was not accompanied by any significant changes in liver/body weight ratios, liver morphology or induction of hepatic CYP1A1-dependent activity which is one of the most sensitive indicators of exposure to AhR agonists. RT-PCR and Western blot analysis of mammary tumor mRNA and protein extracts, respectively, confirmed the presence of AhR suggesting that AhR-mediated signaling pathways are functional in rat mammary tumors. These results define a relatively non-toxic group of AhR agonists which exhibit potent antitumorigenic activity in the DMBA-induced rat mammary tumor model (<1 mg/kg/day), and therefore represent a new class of indirect-acting antiestrogens which have potential for clinical treatment of mammary cancer.

Drug and poison information resources on the Internet, Part 2: Identification and evaluation.

The Internet is a potential source of information for practitioners and researchers of pharmaceutical sciences. Despite its explosive growth and popularity, pharmacists and other health care providers have been slow to use the Internet as a source of clinical information. We have identified and evaluated several sites available on the Internet that are devoted to providing information related to professional practice, pharmacotherapy, and toxicology. We have concerns, however, regarding the quality of the available information and advise users to be cautious in using the data they obtain.

Vancomycin pharmacokinetics and dosing in premature neonates.

We prospectively studied the pharmacokinetic parameters of vancomycin in premature neonates given vancomycin according to a dosage protocol developed in our neonatal unit. Study infants were administered vancomycin according to four postconceptional age (PCA) groups: (0) 18 mg/kg every 36 h for PCA < 27 weeks; (I) 16 mg/kg every 24 h for PCA 27-30 weeks; (II) 18 mg/kg every 18 h for PCA 31-36 weeks; and (III) 15 mg/kg every 12 h for PCA > or = 37 weeks. Pharmacokinetic parameters were calculated from peak and trough serum vancomycin concentrations at steady state. Results in 44 infants (PCA, 27-44 weeks) showed that our dosage regimen achieved target peak serum vancomycin concentrations in 64% of neonates in Groups I-III, although it tended to undershoot the target trough concentrations. Volume of distribution (Vd), normalized for body weight, remained constant throughout the PCA range, with a mean value of 0.56 L/kg, whereas absolute clearance (r = 0.81) and normalized clearance (r = 0.48) increased with PCA (p < 0.005). The increase in clearance with PCA is associated with a greater elimination rate constant and shorter half-life. Vancomycin therapy can be initiated in a standard fashion according to our protocol or by individualizing the dosage regimen based on a Vd of 0.56 L/kg and clearance estimated from the infant's body weight and PCA groups.

In vitro assessment of vancomycin HCl compatibility after coinfusion with a specialized amino acid formulation.

Vancomycin usage at British Columbia's Children's Hospital has increased substantially in the Special Care Nursery as a consequence of a study demonstrating a reduced morbidity and mortality in neonates with necrotizing enterocolitis when treated with vancomycin and cefotaxime. The inability to place more than one peripheral intravenous access necessitates interruption of parenteral nutrition to infuse vancomycin, resulting in a reduction of the planned daily intake of these neonates. This is clinically significant with the administration of vancomycin because of the long administration period required for this drug (60 minutes). This study was designed to assess the physical and chemical stability of vancomycin with a standard neonatal parenteral nutrition solution, Vamin A, when coadministered through the same intravenous line. To simulate the actual clinical setting, the dose of vancomycin and the infusion rate of Vamin A were chosen to represent those commonly used in a 1-kg neonate. Physical compatibility was assessed using effluent obtained after coinfusion of vancomycin with parenteral nutrition solution. Duplicate samples were visually checked for color changes and precipitate. High-pressure liquid chromatography (HPLC) and pH testing were used to assess chemical compatibility of vancomycin. The results of physical compatibility revealed no color change or precipitate. No changes in pH were observed. HPLC determination confirmed that there were no significant time-dependent changes in vancomycin stability. The samples were studied over 24 hours to determine the rate of degradation of vancomycin, if any, under various temperature conditions. The concentrations were not significantly different from each other at the different temperatures studied. Thus, there was no apparent change in the concentration of vancomycin in the presence of Vamin A.(ABSTRACT TRUNCATED AT 250 WORDS)