afimoxifene and alpha-naphthoflavone

Polychlorinated biphenyls (PCBs) have been detected at high levels, up to hundreds of pg/ml, in human ovarian follicle fluid. The effect of PCBs on the ovary and the consequences of exposure are largely unknown. We have previously shown that PCB3 (4-chlorobiphenyl) increases the secretion of estradiol and the activity of cytochrome P450s (CYPs) in ovarian follicle cells. Our goal here is to elucidate the mechanism of CYP induction by this congener. Exposure of porcine follicle cells, a co-culture of theca and granulosa cells, to 6 ng/ml of PCB3 caused an increase in CYP1A1 protein and enzymatic activity, in the same manner as exposure to exogenous 17beta-estradiol. No changes were seen in the protein level of the aryl hydrocarbon receptor (AhR), which mediates the first step in the signaling pathway of CYP1A1 induction. However, a strong reduction was seen in the protein level of estrogen receptor beta (ERbeta), while no effect was seen on ERalpha protein levels. These result suggest that: 1) PCB3 acts as an agonist of ERbeta but not the Ah receptor in the ovarian follicles, 2) PCB3 is not only an efficacious inducer of CYP1A1 expression and activity, but also a substrate for this enzyme. Changes in the expression level of CYP1A1 not only alter the intensity of the activity of PCB3, but also the activity of estrogen in the ovary.

Topics: Animals; Benzoflavones; Biphenyl Compounds; Blotting, Western; Coculture Techniques; Cytochrome P-450 CYP1A1; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation, Enzymologic; Granulosa Cells; Ovarian Follicle; Ovary; Receptors, Aryl Hydrocarbon; Swine; Tamoxifen; Theca Cells

The formation of reactive oxygen species (ROS) plays a critical role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicities in mammalian cells since it promotes cell proliferation, growth arrest, and apoptosis. In this study, we investigated whether TCDD induces oxidative stress and DNA damage in human ERalpha(+)/MCF-7 and ERalpha(-)/MDA-MB-231 breast cancer cells and whether this is accompanied by the initiation of DNA repair events. Results indicated that viability of MCF-7 and MDA-MB-231 cells was concentration- and time-dependently reduced by TCDD. Further, we observed significant increases in ROS formation and decreases in intracellular glutathione (GSH) in these two cell lines after TCDD treatment. Overall, the extent of cell death was greater in MCF-7 cells than in MDA-MB-231 cells whereas the magnitude of ROS formation and GSH depletion was greater in MDA-MB-231 cells than in MCF-7 cells. In addition, we observed that at non-cytotoxic concentration (1nM for 5h), TCDD induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 and MDA-MB-231 cells. These decreases were completely blocked by three types of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. The catalytic activation of PARP-1 in cells treated with TCDD was confirmed by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Moreover, we demonstrated increases in the number of DNA strand breaks in MCF-7 and MDA-MB-231 cells exposed to TCDD as measured by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that TCDD induces decreases in intracellular NAD(P)H and NAD(+) through PARP-1 activation mediated by formation of DNA strand breaks. In addition, we demonstrated that the extent of oxidative stress and DNA damage was greater in MDA-MB-231 cells than in MCF-7 cells, with a strong correlation to estrogen receptor (ER) status. In conclusions, our findings add further support to the theme that ROS formation is a significant determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to TCDD and that the TCDD-induced oxidative stress and DNA damage may, in part, contribute to TCDD-induced carcinogenesis.

Topics: Benzamides; Benzoflavones; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Survival; Coumarins; DNA Breaks; Environmental Pollutants; Estrogen Receptor alpha; Glutathione; Humans; NAD; NADP; Oxidative Stress; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Polychlorinated Dibenzodioxins; Reactive Oxygen Species; Receptors, Aryl Hydrocarbon; Tamoxifen

The primary purpose of this research is to investigate whether exposure to polychlorinated biphenyls (PCBs), i.e. PCB153 and PCB126, is associated with induction of reactive oxygen species (ROS), poly(ADP-ribose) polymerase-1 (PARP-1) activation, and cell death in human T47D and MDA-MB-231 breast cancer cells. Results indicated that PCB153 and PCB126 induced concentration- and time-dependent increases in cytotoxic response and ROS formation in both T47D and MDA-MB-231 cells. At non-cytotoxic concentrations both PCB153 and PCB126 induced decreases in intracellular NAD(P)H and NAD+ in T47D and MDA-MB-231 cells where T47D cells were more resistant to PCB-induced reduction in intracellular NAD(P)H than MDA-MB-231 cells. Further investigation indicated that three specific PARP inhibitors completely blocked PCB-induced decreases in intracellular NAD(P)H in both T47D and MDA-MB-231 cells. These results imply that decreases in intracellular NAD(P)H in PCB-treated cells may be, in part, due to depletion of intracellular NAD+ pool mediated by PARP-1 activation through formation of DNA strand breaks. Overall, the extent of cytotoxic response, ROS formation, and PARP-1 activation generated in T47D and MDA-MB-231 cells was greater for PCB153 than for PCB126. In addition, the cytotoxicity induced by PCB153 and PCB126 in both T47D and MDA-MB-231 cells was completely blocked by co-treatment of catalase, dimethylsulfoxide, cupper (I)-/iron (II)-specific chelators, and CYP1A/2B inhibitors. This evidence suggests the involvement of ROS, Cu(I), Fe(II), and CYP1A/2B enzymes in mediating the induction of cell death by PCB153 and PCB126. Further, antagonism was observed between PCB126 and PCB153 for effects on cytotoxic response and ROS formation in T47D and MDA-MB-231 cells. Antagonism was also observed between PCB153 and PCB126 in the induction of NAD(P)H depletion at lower concentration (<10 microM) in T47D cells, but not in MDA-MB-231 cells. In conclusions, results from our investigation suggest that ROS formation induced by PCBs is a significant determinant factor in mediating the DNA damage and cell death in human breast cancer cells. The data also suggests that the status of estrogen receptor alpha may play a role in modulating the PCB-induced oxidative DNA damage and cell death in human breast cancer cells.

Topics: Benzoflavones; Breast Neoplasms; Catalase; Cell Line, Tumor; Cell Survival; Chelating Agents; Cytochrome P-450 Enzyme Inhibitors; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Female; Fluoresceins; Humans; Metyrapone; Molecular Structure; NAD; NADP; Oxidation-Reduction; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Polychlorinated Biphenyls; Reactive Oxygen Species; Tamoxifen

Tamoxifen has been found to be metabolized by liver primarily into three metabolites, tamoxifen-N-oxide, formed by the flavin-containing monooxygenase, and N-desmethyl- and 4-hydroxytamoxifen, formed by cytochrome P450. The N-demethylation was demonstrated to be catalyzed by P4503A in rat and human liver; however, the P450s catalyzing the 4-hydroxylation have not been identified. Although 4-hydroxytamoxifen exhibits more potent estrogen agonist/antagonist activity than tamoxifen, the relative contributions of the parent drug and its 4-hydroxy metabolite(s) to the activity of tamoxifen in vivo have not been established. We report here that the rate of tamoxifen 4-hydroxylation is higher in livers of adult chicken and chick embryos than in livers of mammalian species. Tamoxifen 4-hydroxylation was increased by treatment of chick embryos with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and to a lesser extent by phenobarbital (PB). The major effect of PB treatment was an increase in tamoxifen N-demethylation. Tamoxifen 4-hydroxylase activity of reconstituted purified chicken P450s was highest for TCDDAA, a P450 active in arachidonate epoxygenation and estradiol 2-hydroxylation, and one of the two major P450s induced by TCDD and beta NF in chick embryo liver. The second P450, TCDDAHH, which is active in aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase was inactive in tamoxifen 4-hydroxylation. Anti-TCDDAA IgG immunoinhibited tamoxifen 4-hydroxylation in microsomes from beta NF-treated embryos by over 80%, but was ineffective against this reaction in the controls. The immunochemical findings together with the reconstitution data identify TCDDAA as the P450 responsible for TCDD/beta NF-induced tamoxifen 4-hydroxylation in chick liver. In PB-treated livers, a P450 fraction containing CYP2H1/H2, the major PB-induced P450s, had the highest tamoxifen 4-hydroxylase and N-demethylase activities, a finding compatible with one or both of those P450s being responsible for the PB-induced tamoxifen 4-hydroxylation and N-demethylation. The findings reported here raise the possibility that exposure of women undergoing tamoxifen therapy to agents that induce human CYP1A2 or CYPB1/2 analogues may produce increased levels of 4-hydroxytamoxifen and that this may affect the therapeutic potency of tamoxifen.

Topics: Animals; Benzo(a)pyrene; Benzoflavones; beta-Naphthoflavone; Chick Embryo; Chickens; Cricetinae; Cytochrome P-450 Enzyme System; Enzyme Induction; Estrogen Antagonists; Female; Humans; Hydroxylation; Immunoglobulin G; Liver; Mesocricetus; Mice; Microsomes, Liver; Models, Biological; Phenobarbital; Polychlorinated Dibenzodioxins; Rats; Rats, Sprague-Dawley; Species Specificity; Tamoxifen