Harold E. Seifried

Metabolism of benzo[a]pyrene. Effect of 3-methylcholanthrene pretreatment on metabolism by microsomes from lungs of genetically "responsive" and "nonresponsive" mice.

Abstract The metabolism of [14C]benzo[a]pyrene by microsomes from the lungs of normal and 3-methylcholanthrene-treated DBA/2J, C57BL/6J, and A/HeJ mouse strains was quantitatively analyzed by high-pressure liquid chromatography. The ratio of dihydrodiols of benzo[a]pyrene to total metabolites formed was greater with lung microsomes than with liver microsomes in all three strains. The ratio of epoxide hydrase to monooxygenase activity in mouse lung was shown to be considerably higher than in mouse liver. Benzo[a]pyrene metabolism by control lung microsomes showed some strain differences. C57BL/6J and A/HeJ mice formed twice as much dihydrodiols as a percentage of total metabolism compared to DBA/2J mice. DBA/2J mice produced somewhat less phenol 2 fraction and considerably more quinone 1 and 2 fractions than the other two mouse strains as a percentage of total metabolism. Treatment of C57BL/6J and DBA/2J mice with 3-methylcholanthrene resulted in a 20-fold increase in the metabolism of benzo[a]pyrene, while A/HeJ mice were induced more than 50-fold. The profiles of metabolites from the 3-methylcholanthrene-induced animals were nearly identical in all three mouse strains.

Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells.

The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in Salmonella typhimurium and L5178Y mouse lymphoma cells. Except for the weak response obtained with ferrous fumarate, none of the compounds induced a mutagenic response in Salmonella. In the mouse lymphoma assay, responses were related to the Fe compound and/or reduction of ferric (Fe+3) to ferrous (Fe+2). Responses with the elemental forms of Fe were divergent. Electrolytic Fe with a relatively larger particle size and irregular shape was negative. The smaller‐sized carbonyl Fe, which after 4 hr attached to and was taken up by the cells, induced mutagenic responses both with and without S9. With ferric chloride (FeCl3) and ferric phosphate (FePO4), there was an increase in mutant frequency only with S9. With the Fe+2 compounds, ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), positive responses were observed without S9. The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9. The lowest effective doses (LED) for induction of mutagenicity were identified for these compounds and an LED ratio calculated. The LED ratio ranges from 1 for FeSO4 to 30 for carbonyl Fe, which are similar to oral LD50 values obtained in animal studies. Environ. Mol. Mutagen. 33:28–41, 1999

The 15kDa Selenoprotein and Thioredoxin Reductase 1 Promote Colon Cancer by Different Pathways

Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

Dietary Selenium Levels Affect Selenoprotein Expression and Support the Interferon-γ and IL-6 Immune Response Pathways in Mice.

Selenium is an essential element that is required to support a number of cellular functions and biochemical pathways. The objective of this study was to examine the effects of reduced dietary selenium levels on gene expression to assess changes in expression of non-selenoprotein genes that may contribute to the physiological consequences of selenium deficiency. Mice were fed diets that were either deficient in selenium or supplemented with selenium in the form of sodium selenite for six weeks. Differences in liver mRNA expression and translation were measured using a combination of ribosome profiling, RNA-Seq, microarrays, and qPCR. Expression levels and translation of mRNAs encoding stress-related selenoproteins were shown to be up-regulated by increased selenium status, as were genes involved in inflammation and response to interferon-γ. Changes in serum cytokine levels were measured which confirmed that interferon-γ, as well as IL-6, were increased in selenium adequate mice. Finally, microarray and qPCR analysis of lung tissue demonstrated that the selenium effects on immune function are not limited to liver. These data are consistent with previous reports indicating that adequate selenium levels can support beneficial immune responses, and further identify the IL-6 and interferon-γ pathways as being responsive to dietary selenium intake.

Selenophosphate synthetase 1 is an essential protein with roles in regulation of redox homoeostasis in mammals

Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogues, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyses the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1-knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by embryonic day (E)8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione transferase Omega 1 (GSTO1). To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma (EC) cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Furthermore, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homoeostasis and controlling cell growth.

Anthracene 1,2-oxide: synthesis and role in the metabolism of anthracene by mammals

Synthesis of anthracene 1,2-oxide proceeds in good yield via dehydrobromination of 1 -acetoxy-2,4-dibromo1,2,3,4-tetrahydroanthracene. In contrast, dehydrobromination of 2-acetoxy-3,4-dibromo-1,2,3,4-tetrahydroanthracene produced 1 -bromoanthracene instead of the desired anthracene 2,3-oxide or its oxepin tautomer. Nucleophilic attack of thioethoxide on anthracene 1,2-oxide yields trans-1 -hydroxy-2-ethylthio-1,2-dlhydroanthracene which readily eliminates water with concomitant migration of the thioethoxide group to produce 1 - anthryl ethyl sulphide. In vitro metabolism of anthracene with liver microsomes forms predominantly trans-1,2-dihydroxy-l,2-dihydroanthracene with little evidence of metabolism at the 9,10-position.

Chapter 16 – Antioxidants in Health and Disease

Antioxidant compounds comprise a myriad of structures with functions in addition to their well-known roles in scavenging reactive oxygen species, eliminating free radicals, and preventing oxidative damage. Studies to elucidate the efficacy and mechanism(s) of action of several specific antioxidants on prevention and treatment of cancer and cardiovascular disease have had mixed success. The overall value of dietary antioxidants notwithstanding, confusion regarding appropriate dosage and administration forms has led to inconclusive results in many cases. The existence of J- or U-shaped dose–response curves, with negative effects being observed at both low and high doses, has also complicated the study of these compounds. The importance of maintaining a homeostatic level of oxidative stress and the occurrence of large numbers of active antioxidants in the diet were only recently fully appreciated. Also, recent awareness of variable genetic susceptibility to both the positive and the negative effects, as well as potential interactions with cancer treatment regimens, supports the need for further study in this area.

Broccoli consumption affects the human gastrointestinal microbiota

The human gastrointestinal microbiota is increasingly linked to health outcomes; however, our understanding of how specific foods alter the microbiota is limited. Cruciferous vegetables such as broccoli are a good source of dietary fiber and phytonutrients, including glucosinolates, which can be metabolized by gastrointestinal microbes. This study aimed to determine the impact of broccoli consumption on the gastrointestinal microbiota of healthy adults. A controlled feeding, randomized, crossover study consisting of two 18-day treatment periods separated by a 24-day washout was conducted in healthy adults (n=18). Participants were fed at weight maintenance with the intervention period diet including 200 g of cooked broccoli and 20 g of raw daikon radish per day. Fecal samples were collected at baseline and at the end of each treatment period for microbial analysis. Beta diversity analysis indicated that bacterial communities were impacted by treatment (P=.03). Broccoli consumption decreased the relative abundance of Firmicutes by 9% compared to control (P=.05), increased the relative abundance of Bacteroidetes by 10% compared to control (P=.03) and increased Bacteroides by 8% relative to control (P=.02). Furthermore, the effects were strongest among participants with body mass index <26 kg/m2, and within this group, there were associations between bacterial relative abundance and glucosinolate metabolites. Functional prediction revealed that broccoli consumption increased the pathways involved in the functions of the endocrine system (P=.05), transport and catabolism (P=.04), and energy metabolism (P=.01). These results reveal that broccoli consumption affects the composition and function of the human gastrointestinal microbiota.

Workshop Report: Modulation of Antitumor Immune Responses by Dietary and Microbial Metabolites

The human microbiota maintains an enormous and diverse capacity to produce a diet-dependent metabolome that impacts both host tissue and microbial community homeostasis. Recent discoveries support a growing appreciation that microbial metabolites derived from bioactive foods are also important regulators of host immune and metabolic functions. To gain a better understanding of the current evidence for the roles of dietary and microbial metabolites in tumor immunity, the Division of Cancer Biology and the Division of Cancer Prevention, National Cancer Institute, cosponsored a workshop on August 31 and September 1, 2016, in Bethesda, Maryland. Workshop participants examined several lines of converging science that link nutrition, microbiology, and tumor immunology and identified key concepts and research opportunities that will accelerate our understanding of these interactions. In addition, the participants identified some of the critical gaps and research challenges that could be addressed through interdisciplinary collaborations, including future opportunities for translating new information into novel cancer prevention and treatment strategies based on targeting host immune functions that are altered by metabolite sensing pathways.

Abstract 824: Effects of metfornin on mammary carcinogenesis and metabolic profiles in methylnitrosourea-treated rats on standard or Western diets

The methylnitrosourea-induced model of ER + mammary cancer in female Sprague-Dawley rats has been routinely used to screen for chemopreventive agents. We recently reported that metformin was ineffective in reducing cancer formation in rats on a standard (4% fat) diet (Thompson, et al., Cancer Prev Res. 8, 231, 2015). In the present studies, rats were placed on a standard (Teklad) diet or on a Western diet (20% fat, low calcium) at 43 days of age (DOA), and administered MNU at 50 DOA. Rats were administered metformin or vehicle daily by gavage beginning at 57 days of age. Serum from the rats of the various groups was obtained both at 21 days following initial administration of metformin (or vehicle) and at the end of the study (when tumors had developed in most rats). We found an increase in the final body weights, number of mammary cancers, and the final weights of tumors in rats on western diet compared with standard diet. Furthermore, metformin failed to decrease tumor multiplicity in either diet, in fact, it increased final cancer weights (P Citation Format: Mark S. Miller, Matthew D. Thompson, Ronald A. Lubet, Vernon E. Steele, Harold E. Seifried, Clinton J. Grubbs. Effects of metfornin on mammary carcinogenesis and metabolic profiles in methylnitrosourea-treated rats on standard or Western diets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 824.