Glucosinolates have been implicated as a mediator of the cancer-protective properties of cruciferous vegetables. Enzymatic hydrolysis of glucosinolates by plant or microbial myrosinase yields a range of metabolites including beneficial isothiocyanates. Little is known about the fate of glucosinolates after their ingestion. Using urinary end-products of metabolism as markers, measurement of the production of isothiocyanates in the digestive tract of monogastric animals has been achieved. Initially, a range of isothiocyanates were administered to rats and their excretion as mercapturic acids was quantified. Relative recovery of different isothiocyanates was found to be consistent and predictable, allowing the use of artificial isothiocyanates as recovery standards in subsequent experiments. Subsequently, the relative influence of plant and bacterial myrosinase on isothiocyanate production was quantified in rats. A proportion of 0.80 (s. e. m. 0.076) of benzyl glucosinolate was hydrolysed to isothiocyanate by plant myrosinase. In the presence of both plant and microbial activity,, the proportion of benzyl isothiocyanate release was significantly decreased (0.50 s. e. m. 0.046, p<0.01) suggesting microbial breakdown of isothiocyanates. The approach, adapted for use with human subjects showed that the proportions of allyl isothiocyanate measured after ingestion of raw and cooked cabbage were 0.37 (s. e. m. 0.045) and 0.53 (s. e. m. 0.134) respectively in healthy male volunteers. A further experiment with rats established that isothiocyanate uptake in the distal digestive tract was significantly less than in the proximal intestine (0.12 s. e. m. 0.017 and 0.48 s. e. m. 0.029 respectively), suggesting a potential underestimation of isothiocyanate release in the distal digestive tract when using urinary markers. Finally, enhancement of bacterial fermentation by addition of inulin to the diet had little influence on isothiocyanate production in the gut. The findings suggested that the formation of the cancer-protective isothiocyanates was significant, in vivo, thereby strengthening the evidence for a beneficial effect of cruciferous vegetables for health. The newly developed method opens up possibilities of concurrently exploring the digestive fate of isothiocyanates and the toxicity of carcinogenic compounds.

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Acute lymphoblastic leukemia (ALL) encompasses a spectrum of lymphoid progenitors that have undergone malignant transformation and clonal proliferation at various stages of differentiation. Some cases of ALL have been documented to have prenatal origins and in particular neonatal exposure to various environmental pollutants is associated with increased disease risk, including childhood lymphoma and leukemia. Dibenzo[def,p]chrysene (DBC) is a polycyclic aromatic hydrocarbon (PAH) and in our laboratory has been established as a transplacental carcinogen in mice, producing aggressive T-cell lymphoblastic lymphomas, lung, liver, uterine, ovarian, and testicular lesions, depending on timing and dose of exposure. Investigation of the transplacental and translactational transfer of DBC was warranted following a cross-foster experiment demonstrating the greatest tumorigenic response occurred in offspring both gestating in and nursed by an exposed female. [¹⁴C]-DBC (GD17) dosing was utilized to examine time-dependent alterations of [¹⁴C] in maternal and fetal tissues, excreta, and residual levels at weaning. Fetal tissue levels of [¹⁴C]-DBC equivalents were 10-fold lower than maternal tissue, and after weaning the residual body burden was roughly equivalent in offspring exposed only in utero or only via lactation. Certain bioactive food components, including indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), and sulforaphane (SFN) from cruciferous vegetables have been shown to target cellular pathways regulating carcinogenesis. In the above mentioned DBC initiated model of carcinogenesis, I3C is an effective transplacental chemopreventive agent. We sought to extend our chemoprevention studies in mice to a human neoplasm in cell culture, analogous to the observed murine T-cell lymphomas. Treatment of the human T-ALL cell line CCRF-CEM (CEM) with I3C reduced cell proliferation and viability only at supraphysiologic concentrations whereas DIM, the primary acid condensation product of I3C, had a marked effect at low micromolar concentrations in vitro and reduced growth of CEM xenografts in vivo. Additional T-ALL lines, selected to represent the heterogeneity of the disease, (CCRF-HSB2, Jurkat, and SUP-T1) responded similarly in vitro, demonstrating a potential therapeutic value of DIM in T-ALL. Given that epigenetic reprograming is especially active during fetal development and that DNA hypermethylation contributes to the etiology of T-ALL we examined genome-wide DNA methylation in CEM. Differential methylation analysis revealed that DIM and I3C alter CpG methylation in unique, yet overlapping, gene targets. DIM treated cells exhibited a dose-dependent decrease in hypermethylation, an observation consistent with an epigenetic mechanism of cancer suppression. Pyroseqencing and RTPCR technologies were utilized to validate changes in DNA methylation and to compare these patterns with a transcriptional response in both novel targets and candidate genes selected from the literature. Collectively, these studies merited returning to the murine transplacental model for further investigation of genetic and epigenetic changes upon maternal dietary intervention with I3C. More importantly we incorporated whole cruciferous vegetable diets (10% broccoli sprouts or 10% Brussels sprouts), SFN diet, or the combination of SFN and I3C, in order to examine matrix and mixture effects. Preliminary analysis suggests a worse prognosis for those animals exposed in utero to SFN or the whole foods, especially males. As this is the first study to administer SFN or whole cruciferous vegetables in a transplacental model of carcinogenesis, our results warrant further study on the concentration dependent influence of these potent phytochemicals during the perinatal window. / Graduation date: 2012