Early cellular events in apoptosis of pituitary cells during exposure to PCBs.

<p>Primary pituitary cells were incubated for 24 hours with fresh serum-free medium containing 10 μM PCBs or vehicle (DMSO 0.1%). Apoptosis was evaluated by flow cytometric analysis after co-staining of the pituitary cells with annexin V-FITC and propidium iodide (PI), as detailed in the Materials and Methods. (A) Representative flow cytometric scatter plots of Annexin V staining assay: the right bottom quadrant of each plot contains the early apoptotic cells (annexin V-FITC positive/PI negative). The PCB mixture Aroclor 1254 (ARO) induced an early phase of pituitary cell apoptosis. Two non-dioxin-like congeners reduced (PCB 153) or increased (PCB 180) apoptosis, respectively. In contrast, two dioxin-like congeners (PCB 126 and PCB 77) did not significantly affect apoptosis. Cisplatin (32 μM) was used as a positive control. The scatter plot for untreated cells is also reported. (B) Percentages of annexin V-FITC positive cells in the different treatments. Results represent the mean value ± SD of five independent experiments. **p<0.001, ***p<0.0001 compared with the vehicle, according to ANOVA followed by Dunnett’s post hoc test.</p

The 5-bromo-2’-deoxyuridine uptake.

<p>Cell proliferation was evaluated by measuring the cellular uptake of 5-bromo-2’-deoxy-uridine, in the presence of individual congeners (PCB 153, PCB 180, PCB 126, PCB 77) or the Aroclor 1254 mixture (ARO), by BrdU incorporation ELISA colorimetric assay, as detailed in the Materials and Methods. Primary pituitary cells were incubated for 24 hours with fresh serum free-medium containing the test substances at 10 μM concentration or with vehicle (DMSO 0.1%). The culture medium, supplemented with 10% fetal bovine serum and Colchicine (1 μM) were used as a positive or negative control, respectively. Only in the presence of the non-dioxin-like congener PCB 153, did the uptake of 5-bromo-2’-deoxy-uridine increase, suggesting the potential growth of pituitary cells exposed to this congener. Data represent the mean ± SD of five independent experiments, each performed in quadruplicate and expressed as arbitrary units (A.U.), assuming that the BrdU incorporation level obtained from the cells exposed to vehicle was considered as 1. **, p<0.001, ***, p<0.0001 compared with vehicle, according to ANOVA followed by Dunnett’s post hoc test.</p

Effects of PCBs exposure on the molecular mechanism of apoptosis.

<p>Representative Western blot from pituitary primary cell extracts obtained as described in the Materials and Methods. Primary pituitary cells were incubated for 24 hours with fresh serum-free medium containing the test substances at a 10 μM concentration or the vehicle (DMSO 0.1%). The expression of both the cytochrome c, as regulator of caspase-9 (intrinsic mechanism), and TRADD, as regulator of caspase-8 (extrinsic mechanism) were evaluated (A, B). Cisplatin (32 μM) or TNFα (10 μM) were used as positive controls for cytochrome c or TRADD, respectively. In line with the results on caspases expression and activity, both mechanisms were hindered by PCB 153 treatment. TRADD expression also increased by cell exposure to Aroclor 1254 or PCB 180, while cytochrome c expression remained unaltered. Data are expressed as arbitrary units (A.U.), which represent the ratio between the intensity of the band of interest and the intensity of the band corresponding to the control protein (β-actin). The mean ± SD of measurements obtained in five independent experiments are reported. **, p<0.001, ***, p<0.0001 compared with vehicle, according to ANOVA followed by Dunnett’s post hoc test.</p

Proposed mechanism regulating the apoptotic pathway by PCBs in pituitary cells.

<p>The non-dioxin-like PCB 180 increased apoptosis by activating the extrinsic pathway (TRADD, caspase-8). In contrast, the extrinsic (TRADD, caspase-8) and intrinsic (cytochrome c, caspase-9) pathways were down-regulated by the non-dioxin-like PCB 153, leading to an anti-apoptotic and putative proliferative phenotype. Changes in apoptosis by PCBs occur through the interference of PCB with TR, AhR and CYP1A1 action. A proposed mechanism is shown featuring the disruption of TR action by PCB 153 after its hydroxilation by CYP1A1, leading to an anti-apoptotic phenotype. In contrast, the disruption of AhR by PCB 180 led to a pro-apoptotic phenotype.</p

Changes in late phase of pituitary cells apoptosis during exposure to PCBs.

<p>The evaluation was performed by measuring the cellular DNA fragmentation level using the ELISA colorimetric test (A) or terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay (B, C), as in the Materials and Methods. Cisplatin (32 μM) was used as a positive control. A late phase of pituitary cells apoptosis was raised by Aroclor 1254 or PCB 180, was almost suppressed by PCB 153 or left unaltered by the dioxin-like congeners PCB 126 and PCB 77. (A) Primary pituitary cells were incubated for 24 hours with fresh serum-free medium containing 10 μM PCBs or vehicle (DMSO 0.1%). Data represent the mean values ± SD of five independent experiments, each performed in quadruplicate and are expressed as arbitrary units (A.U.), assuming that the DNA fragmentation level obtained from the cells exposed to vehicle was considered as 1. **, p<0.001, ***, p<0.0001, compared with vehicle, according to ANOVA followed by Dunnett’s post hoc test. (B, C) Primary pituitary cells were treated as described in the study design. Specificity of TUNEL (green) nuclear labelling was confirmed by DAPI (blue), which selectively identifies nuclei. Merged pictures represent cells stained with both TUNEL and DAPI. Results represent the mean ± SD of five independent experiments and are expressed as arbitrary units (A.U), considering the TUNEL level obtained from the cells exposed to vehicle as 1. *, p<0.01, ***, p<0.0001 compared with vehicle, according to ANOVA followed by Dunnett’s post hoc test. Magnification, 40X; bars = 50μm.</p

Evaluation of apoptosis induced by ROS production.

<p>Quantitative ROS generation was measured using the oxidant-sensitive probe dichlorofluorescein (H2DCF-DA) assay (A), apoptosis was evaluated by ELISA DNA fragmentation assay (B), and caspase-3 activity was determined by fluorimetric assay (C), as described in the Materials and Methods. Pituitary primary cells were treated as described in the study design. 10 μM Hydrogen Peroxide (H₂O₂) (A), 50 μM H₂O₂ (B, C), or 32 μM Cisplatin (B,C) were used as positive controls. In all the assays, 1 mM Ascorbic Acid (vitamin C) was used as an antioxidant. ROS production was not influenced by non-dioxin-like PCB 180 or PCB 153, with or without the addition of vitamin C (A). Moreover, vitamin C had no effect on either apoptosis or caspase-3 activity of PCBs-treated pituitary cells (B, C), suggesting that ROS do not play a key role in PCBs-mediated apoptosis mechanism. Results represent the mean ± SD of five independent experiments, each performed in quadruplicate and expressed as arbitrary units (A.U.). The value of 1 was assigned to ROS production (A), DNA fragmentation (B) or caspase-3 activity (C) from vehicle-exposed cells. **, p<0.001, ***, p<0.0001 compared with vehicle, according to ANOVA followed by Dunnett’s post hoc test. #, p<0.05, ##, p<0.005, ###, p<0.0005, p = n.s. (not significant) according to Student’s t-test adjusted for multiple comparisons following the Bonferroni method.</p

Changes in the activity of caspases during exposure to PCBs.

<p>Caspase activity was assessed by fluorimetric or luminometric assays, as described in the Materials and Methods. Cisplatin (32 μM) was included as a positive control. The activity of the three individual caspases decreased by exposure to PCB 153 (A, B, C). On the other hand, the activity of both caspase-3 and caspase-8 increased after treatment with Aroclor 1254 or PCB 180 (A, B), while the activity of caspase-9 was unaffected (C). To confirm the specificity of PCB action on caspases, cells were also pre-treated with caspase inhibitors prior to PCB exposure. 20 μM Ac-DEVD-CHO (A), Ac-IETD-CHO (B) or Ac-LEHD-CHO (C) were used as inhibitors of caspase -3, -8 or -9, respectively. Data (mean ± SD of five independent experiments) were expressed as arbitrary units (A.U.); the value of 1 was attributed to the caspase activity of vehicle-exposed cells. **, p<0.001, ***, p<0.0001, compared with the vehicle, according to ANOVA followed by Dunnett’s post hoc test. ##, p<0.005, ###, p<0.0005, p = n.s. (not significant), according to Student’s t-test adjusted for multiple comparisons following the Bonferroni method.</p

Changes in the expression of caspases during exposure to PCBs.

<p>Representative Western blot from pituitary primary cell extracts obtained as described in the Materials and Methods. Primary pituitary cells were incubated for 24 hours with fresh serum-free medium containing the test substances at a 10 μM concentration or the vehicle (DMSO 0.1%). Cisplatin (32 μM) was used as a positive control. The expression of the final effector of apoptosis, i.e. caspase-3, changed in relation to the change in apoptosis during the exposure to a mixture of PCBs or individual congeners (A). In addition, the intrinsic (i.e., mithochondrial) apoptotic pathway was not influenced by either the mixture (Aroclor 1254) or the congener PCB 180, as shown by the unchanged expression of caspase-9 (C). In contrast, the non-dioxin-like PCB 180 and Aroclor 1254 increased the expression of caspase-8 (B), suggesting the involvement of the extrinsic pathway. Interestingly, the non-dioxin-like PCB 153 reduced the expression of both caspase-8 and 9 (B, C). Data are expressed as arbitrary units (A.U.), which represent the ratio between the intensity of the band of interest and the intensity of the band corresponding to the control protein (β-actin). The mean ± SD of measurements obtained in five independent experiments, are reported. *, p<0.01, **, p<0.001, ***, p<0.0001, compared with the vehicle, according to ANOVA followed by Dunnett’s post hoc test.</p

Clinical characteristics of the patients (probands and whole cohort) according to <i>MEN1</i> genotype.

<p>Clinical characteristics of the patients (probands and whole cohort) according to <i>MEN1</i> genotype.</p

MEN1 manifestations in the groups of F-MEN1, S-MEN1 probands and <i>MEN1</i> mutation-negative and mutation-positive probands.

<p>(<b>A</b>) Association of the main MEN1-related tumors and tumor aggressiveness in probands with F-MEN1 and S-MEN1 syndrome. (<b>B)</b> Association of the main MEN1-related tumors and tumor aggressiveness in patients of the whole cohort with F-MEN1 and S-MEN1 syndrome. (<b>C)</b> Association of the main MEN1-related tumors in <i>MEN1</i> mutation-positive and mutation-negative probands. <b>(D)</b> Detection rate of <i>MEN1</i> gene mutations within each main clinical presentation in <i>MEN1</i> mutation-positive probands with and without family history. Aty-MEN1 refers to atypical MEN1. Statistical significance was determined by Fisher or Chi-square test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.</p