Transplacental Carcinogenesis with Dibenzo[def,p]chrysene (DBC): Timing of Maternal Exposures Determines Target Tissue Response in Offspring

Dibenzo[def,p]chrysene (DBC) is a transplacental carcinogen in mice (15 mg/kg; gestation day (GD) 17). To mimic residual exposure throughout pregnancy, dams received 4 smaller doses of DBC (3.75 mg/kg) on GD 5, 9, 13 and 17. This regimen alleviated the previously established carcinogenic responses in the thymus, lung, and liver. However, there was a marked increase in ovarian tumors (females) and hyperplastic testes (males). [¹⁴C]-DBC (GD 17) dosing revealed transplacental distribution to fetal tissues at 10-fold lower concentrations than in paired maternal tissue and residual [¹⁴C] 3 weeks post dose. This study highlights the importance of developmental stage in susceptibility to environmental carcinogens.This is the author's peer reviewed final manuscript as accepted by the publisher. The published article is copyrighted by Elsevier and can be found here: http://www.elsevier.com/wps/find/journaldescription.cws_home/506050/description#descriptio

Impact of vitamin C supplementation on placental DNA methylation changes related to maternal smoking: association with gene expression and respiratory outcomes

Background: Maternal smoking during pregnancy (MSDP) affects development of multiple organ systems including the placenta, lung, brain, and vasculature. In particular, children exposed to MSDP show lifelong deficits in pulmonary function and increased risk of asthma and wheeze. Our laboratory has previously shown that vitamin C supplementation during pregnancy prevents some of the adverse effects of MSDP on offspring respiratory outcomes. Epigenetic modifications, including DNA methylation (DNAm), are a likely link between in utero exposures and adverse health outcomes, and MSDP has previously been associated with DNAm changes in blood, placenta, and buccal epithelium. Analysis of placental DNAm may reveal critical targets of MSDP and vitamin C relevant to respiratory health outcomes. Results: DNAm was measured in placentas obtained from 72 smokers enrolled in the VCSIP RCT: NCT03203603 (37 supplemented with vitamin C, 35 with placebo) and 24 never-smokers for reference. Methylation at one CpG, cg20790161, reached Bonferroni significance and was hypomethylated in vitamin C supplemented smokers versus placebo. Analysis of spatially related CpGs identified 93 candidate differentially methylated regions (DMRs) between treatment groups, including loci known to be associated with lung function, oxidative stress, fetal development and growth, and angiogenesis. Overlap of nominally significant differentially methylated CpGs (DMCs) in never-smokers versus placebo with nominally significant DMCs in vitamin C versus placebo identified 9059 candidate "restored CpGs" for association with placental transcript expression and respiratory outcomes. Methylation at 274 restored candidate CpG sites was associated with expression of 259 genes (FDR < 0.05). We further identified candidate CpGs associated with infant lung function (34 CpGs) and composite wheeze (1 CpG) at 12 months of age (FDR < 0.05). Increased methylation in the DIP2C, APOH/PRKCA, and additional candidate gene regions was associated with improved lung function and decreased wheeze in offspring of vitamin C-treated smokers. Conclusions: Vitamin C supplementation to pregnant smokers ameliorates changes associated with maternal smoking in placental DNA methylation and gene expression in pathways potentially linked to improved placental function and offspring respiratory health. Further work is necessary to validate candidate loci and elucidate the causal pathway between placental methylation changes and outcomes of offspring exposed to MSDP

3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells

Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL

Growth of human CEM cell xenografts in SCID mice fed DIM or I3C.

<p> <i>Note:</i></p>**<p>, <i>p</i><0.01 as determined by two-way ANOVA with Dunnett's post-hoc test comparisons for significant effect of experimental diet compared to the time-matched control (day 28 values for tumor volume are shown).</p>##<p>, <i>p</i><0.01 or</p>###<p>, <i>p</i><0.001 as determined by one-way ANOVA with Dunnett's post-hoc test comparisons for significant effects of experimental diets compared to control.</p>†<p>Tumor growth rates were modeled by non-linear regression analyses using the exponential growth equation with least-squares fit (Prism 5). Average doubling time (DT) values are shown and were calculated as follows: DT = [(T_o−T_i)×ln2]/ln(V_o/V_i) where <i>T</i>_i and <i>T_o</i> represent the initial and final time points and <i>V_i</i> and <i>V_o</i> represent initial and final tumor volumes. <i>p</i>-values (extra sum of squares F test) are reported for comparison of calculated growth curves for indicated treatments compared to control diet.</p

I3C and DIM reduce proliferation and viability of CEM cells.

<p>Cells were treated with 0 (□), 1.9 (▴), 3.8 (▿), 7.5 (⧫), 15 (○), or 30 (▪) µM DIM (panels A,C) or 0 (□), 15.6 (▴), 31.3 (▿), 62.5 (⧫), 125 (○), 250 (▪), or 500 (⋄) µM I3C (panels B,D) for 24 or 48 hr, then stained with ViaCount reagent for analysis of viable cell concentration and percent viability. Values are the mean fold change in cell proliferation (panels A, B) or percent viability (panels C, D) ± SEM (n = 3 independent experiments) normalized to control cells at 0 hr. **, <i>p</i><0.01 and ***, <i>p</i><0.001, as determined by two-way ANOVA with Bonferroni post-hoc test comparisons for significant effects of DIM treatments at each time point compared to time-matched vehicle control (0.1% DMSO).</p

DIM induces apoptosis in CEM cells as detected by TUNEL.

<p>The <i>In situ</i> cell death detection kit (TUNEL) was applied to fixed CEM cells treated with 0 to 15 µM DIM for 48 hr. (A) Fluorescence images of control (0 µM) and DIM-treated (15 µM) cells were taken at 20× magnification following TUNEL labeling in mounting medium with DAPI. (B) Flow cytometry was used to identify and quantify cells with no, low (open bar) or high (solid bar) intensity staining. **, <i>p</i><0.01 or ***, <i>p</i><0.001 as determined by one-way ANOVA with Dunnett's post-hoc test comparisons for significant effects of DIM treatments within each intensity category as compared to vehicle control (0 µM DIM, 0.1% DMSO).</p

Inhibition of T-ALL cell growth by DIM and I3C.

<p><i>Note</i>: Non-linear regression analyses (four parameters, variable slope) were performed using data generated from each DIM and I3C concentration-response curve generated for each of the four cell lines tested (GraphPad Prism v5.0, San Diego, CA). IC₅₀ values are the concentrations of DIM or I3C required to inhibit cell proliferation or viability by 50% compared to the vehicle control (0.1% DMSO).</p

DIM reduces expression of cell-cycle regulatory proteins.

<p>Following either 12 hr (gray bars) or 24 hr (black bars) treatment with increasing concentrations of DIM, CEM cells were harvested and protein immunoassays were performed for detection of CCND3, CDK4 and CDK6 proteins (three replicate experiments performed). (A) A representative immunoblot is shown for each protein assay. (B) Values shown are average protein expression ± SEM normalized to β-actin, expressed as a percentage difference from time-matched vehicle controls (0.1% DMSO), which were assigned a value of 100%. *, <i>p</i><0.05 or **, <i>p</i><0.01 compared to 0 µM DIM (vehicle control) as determined by one-way ANOVA with Dunnett's post-hoc test for multiple comparisons; overall ANOVA <i>p</i>-values within each time group are indicated in each panel. In some cases where the <i>p</i>-value for the ANOVA was not <0.05, a significant linear trend was evident, as indicated by trend <i>p</i>-values in the figure. Finally, a Student's <i>t-</i>test (###, <i>p</i><0.001) was performed to compare 15 µM DIM to vehicle control for CCND3 expression at 24 hr because high variability observed at the 3.8 µM concentration confounded the ANOVA post-hoc results (overall effect of DIM was significant).</p

Human T-ALL cell lines used in this study.

*<p>ATCC, American Type Tissue Collection; NIH AIDS Research and Reference Reagent Program.</p>a<p>Indicates presence (+) or absence (−).</p>b<p>Indicates relative expression (−, +,++, or +++).</p

DIM induces apoptosis <i>in vivo</i>.

<p>(A) The <i>In situ</i> cell death detection kit (TUNEL) was applied to xenograft sections following exposure to control diet (CTRL), 100 ppm DIM, 500 ppm I3C (I3C-L), or 2000 ppm I3C (I3C-H). Dark staining indicates apoptotic cells, and the scale bar represents 50 µm. (B) Manual and software-assisted counting was performed for xenograft sections as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034975#pone.0034975.s003" target="_blank">File S1</a> to calculate the percentage of positive cells. ***, <i>p</i><0.001 as determined by one-way ANOVA with Dunnett's multiple comparisons post-hoc test.</p