Progress 10/01/12 to 09/30/17
Outputs
Target Audience:New information and conceptual insights resulting from this research were communicated to a variety of stakeholder groups including undergraduate and graduate students, and agricultural professionals involved in research, education and outreach.Lectures, webinars and online offerings were made available to groups of professionals such as registered dieticians, clinical laboratory scientists, and other medical professionals. Findings were also made available to the general public at large through the media. Changes/Problems:There were no major changes/problems in approach. What opportunities for training and professional development has the project provided?This project provided the opportunity for training of undergraduate and graduate students as well as the development of laboratory techniques related to epigenetic changes in gene expression. How have the results been disseminated to communities of interest?Results were disseminated through book chapters, journal articles including newsletters, seminars, public presentations, and conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1. The farnesoid X receptor (FXR) regulates bile acids (BA) metabolism and possesses tumor suppressor functions. The expression of FXR is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. We investigated the influence of APC inactivation on regulation of FXR expression in colonic mucosal cells. We hypothesized that inactivation of APC epigenetically represses NR1H4 (FXR gene name) expression through increased CpG methylation. Normal proximal colonic mucosa, and normal-appearing adjacent colonic mucosa and colon tumors were collected respectively from wild-type C57BL/6J and Apc deficient (ApcMin/+) male mice. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time PCR. In both normal, and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC (siAPC), and HT-29 cells carrying inactivated APC. In ApcMin/ +mice, constitutive CpG methylation of the Fxra3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp, and increased Cox-2, expression in apparently normal adjacent mucosa and colon tumors. The knock-down of APC in HCT-116 cells increased c-MYC, and lowered (~50%) FXR expression, which was further reduced (~80%) by DCA. In human HCT-116, but not HT-29, colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. We concluded that in mouse colonic mucosa and human colon cells loss of APC function may favor silencing of FXR expression through CpG hypermethylation leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer. Goal 2. Only 5-10% of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95% of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy. We evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague-Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Also, we tested in human estrogen receptor (ER)a-negative sporadic UACC-3199 and ERa-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist a-naphthoflavone (aNF) modulated BRCA-1 and ERa expression. Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples. Goal 3.Mammary tumors induced with DMBA had reduced BRCA-1 and ERa expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with aNF rescued BRCA-1 and ERa, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, aNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERa. TNBC exhibited increased basal AhR and BRCA-1 promoter CpG methylation compared to LUM-A, LUM-B, and HER-2-positive breast tumors.
Publications
- Type:
Books
Status:
Published
Year Published:
2016
Citation:
Romagnolo DF, Selmin OI. Co-Editors. Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease. Humana Press/Springer.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Donovan MG, Selmin OI, Doetschman TG, Romagnolo DF. 2017. Mediterranean Diet: Prevention of Colorectal Cancer. Frontiers in Nutrition, Clinical Nutrition, Dec 5;4:59.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2017
Citation:
Donovan MG, Selmin OI, Romagnolo DF. 2017. Prevention by food bioactives in relation to breast cancer subtype: Epigenetic mechanisms. In Press. Epigenetics of Chemoprevention. Elsevier
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Selmin OI, Donovan MG, Papoutsis AJ, Doetschman T, Romagnolo, DF. 2017. Genistein reverses BRCA-1 CpG methylation in human breast cancer cells with activated AhR. Current Developments in Nutrition. May 2017.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Romagnolo DF, Selmin OI. 2017. Mediterranean Diet: challenges and opportunities for improving health and disease prevention. Nutr. Today, Sep;52(5):208-222.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Selmin OI, Daniels KD, Grunwald JT, Ramos SA, Propper CR, Romagnolo DF. 2016. Epigenetics of breast cancer: modifying role of environmental and bioactive food compounds. Mol Nutr Food Res. 60:1310-29.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Romagnolo DF, Jackson KA, Sparks PL, Selmin OI. 2016. Building the Mediterranean Pyramid- PART B: Balancing the plate. In: Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease. Humana Press/Springer, Eds. Romagnolo, Selmin. Chapter 20, pg. 275-288.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Selmin OI, Romagnolo DF. 2016. Building the Mediterranean Pyramid-PART Mediterranean recipes. In: Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease. Humana Press/Springer, Eds. Romagnolo, Selmin. Chapter 19, pg. 261-273.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Donovan MG, Selmin OI, Doetschman TC, Romagnolo DF. 2016. Mediterranean diet, inflammatory bowel diseases and colon cancer. In: Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease. Humana Press/Springer Pubs, Eds. Romagnolo, Selmin. Chapter 14, pg. 181-201
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Selmin OI, Romagnolo APG, Romagnolo DF. 2016. The Mediterranean diet and neurodegenerative diseases. In: Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease. Humana Press/Springer, Eds. Romagnolo, Selmin. Chapter 12, pg. 153-164.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Romagnolo DF, Selmin OI. 2016. Mediterranean diet and lifestyle in a modern world context
In: Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease. Humana Press/Springer, Eds. Romagnolo, Selmin. Chapter 2, pg. 15-26.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Selmin OI, Fang C, Lyon AM, Doetschman TC, Thompson PA, Martinez JD, Smith JW, Lance PM, Romagnolo DF. 2016. Inactivation of adenomatous polyposis coli reduces bile acid/farnesoid X receptor expression through Fxr gene CpG methylation in mouse colon tumors and human colon cancer cells. J Nutr. 146:236-4
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Meyer S, Chibly AM, Burd R, Limesand KH. Insulin-Like Growth Factor-1-Mediated DNA Repair in Irradiated Salivary Glands Is Sirtuin-1 Dependent. J Dent Res. 2017 Feb;96(2):225-232.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Harris Z, Donovan MG, Branco GM, Limesand KH, Burd R. Quercetin as an Emerging Anti-Melanoma Agent: A Four-Focus Area Therapeutic Development Strategy. Front Nutr. 2016 Oct 31;3:48. eCollection 2016.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target audiences include nutrition scientists, undergraduate, graduate and postdoctoral fellows in training; nutrition clinicians; and nutrition practicing professionals; food industry; and representatives of state and federal funding agencies; and the general public at large. Changes/Problems:No major changes to the research strategy were necessary. What opportunities for training and professional development has the project provided?The project has provided opportunities for training of undergraduate students, graduate students and professional development of scientists. How have the results been disseminated to communities of interest?Results were presented at various conferences including: ABRC and Flinn Foundation Awardee Research Conference. Phoenix, AZ, February 24, 2016. FASEB/ASN Meetings, San Diego, CA. April 2-6, 2016. Annual Meetings of the American Association for Cancer Research, New Orleans, LA. April 16-20, 2016. Arizona Cancer Center Retreat. Tucson, AZ, April 8, 2016. Annual Meetings of NACP, Phoenix, AZ. August 18-19, 2016. International Conference on Nutrition and Cancer, Nutrients Beyond Nutrition. Santa Margherita Ligure, Italy, October 7, 2016. Annual Meetings of the American Institute for Cancer Research. Washington, D.C. November 14-16, 2016. What do you plan to do during the next reporting period to accomplish the goals?We will continue our investigations of the effects of AHR activation on regulation of DNA methylation using global methylation assays and in response to dietary antagonists of the AhR; Continue studies of high fat diet and regulation of bile acid homeostasis. Test the effects of isoflavones (i.e. genistein) on BRCA-1 methylation in mammary epithelial cells and animal models.
Impacts
What was accomplished under these goals?
(1) Determine the mechanisms by which dietary bioactive compounds protect against human diseases. Reduced expression of tumor suppressor genes (TSG) increases the susceptibility to breast cancer. However, only a small percentage of breast tumors is related to family history and mutational inactivation of TSG. Epigenetics refers to non-mutational events that alter gene expression. Endocrine disruptors found in foods and drinking water may disrupt epigenetically hormonal regulation and increase breast cancer risk. We have been working hypothesis that agonists of the aromatic hydrocarbon receptor (AHR); bisphenol A (BPA); and arsenic compounds, induce in TSG epigenetic signatures that mirror those often seen in sporadic breast tumors. Conversely, it is hypothesized that bioactive food components that target epigenetic mechanisms protect against sporadic breast cancer induced by these disruptors. Methods and results: 1) overlaps between epigenetic signatures placed in TSG by AHR-ligands, BPA, and arsenic with epigenetic alterations associated with sporadic breast tumorigenesis; and 2) potential opportunities for prevention of sporadic breast cancer with food components that target the epigenetic machinery. Conclusions: Characterizing the overlap between epigenetic signatures elicited in TSG by endocrine disruptors with those observed in sporadic breast tumors may afford new strategies for breast cancer prevention with specific bioactive food components or diet. (2) Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention Background. Previous studies suggested a causative role for agonists of the aromatic hydrocarbon receptor (AhR) in the etiology of BRCA-1-silenced breast tumors, for which prospects for treatment remain poor. Objective. We investigated the regulation of BRCA-1 by the soy isoflavone genistein (GEN) in human estrogen receptor-a (ERa)-positive (MCF-7) and ERa-negative sporadic (UACC-3199) breast cancer cells, respectively with inducible and constitutively active AhR. Methods. In MCF-7 cells, we analyzed the dose- and time dependent effects of GEN and epigallocatechin-3-gallate (EGCG), selected as a prototype dietary DNA methyl-transferase (DNMT) inhibitor, on BRCA-1 expression following AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and in TCDD-washout experiments. We compared the effects of GEN and EGCG on BRCA-1 CpG methylation and cell proliferation. Controls for DNA methylation and proliferation were changes in expression of DNMT-1, and cyclin D1 and p53, respectively. In UACC-3199 cells, we compared the effects of GEN and a-naphthoflavone (aNF, 7,8-benzoflavone), a synthetic flavone and AhR antagonist, on BRCA-1 expression and CpG methylation, cyclin D1, and cell growth. Finally, we examined the effects of GEN and aNF on BRCA-1; AhR-inducible CYP1A1 and CYP1B1; and AhR mRNA expression. Results. In MCF-7 cells, GEN exerted dose- and time-dependent preventative effects against TCDD-dependent downregulation of BRCA-1. After TCDD washout, GEN rescued BRCA-1 protein expression while reducing DNMT-1 and cyclin D1. GEN and EGCG reduced BRCA-1 CpG methylation and cell proliferation associated with increased p53. In UACC-3199 cells, GEN reduced BRCA-1 and ESR1 CpG methylation, cyclin D1, and cell growth while inducing BRCA-1 and CYP1A1. Conclusions: Results suggest preventative effects for GEN and EGCG against BRCA-1 CpG methylation and downregulation in ERa-positive breast cancer cells with activated AhR. GEN and flavone antagonists of the AhRmaybe useful for reactivation of BRCA-1 and ERa via CpG demethylation in ERa-negative breast cancer cells harboring constitutively active AhR. (3) Discover and characterize novel bioactive dietary compounds that have beneficial or adverse effects on human health. The CpG demethylation of BRCA-1 and ESR1 (ERa) by GEN and aNF in breast cancer UACC-3199 cells suggested regimens based on dietary flavonoids may have clinical relevance for therapy of tumors with BRCAness. In support of this inference, we noted that GEN and aNF hampered the expression of cyclin D1 and growth of UACC-3199 cells. aNF is an AhR antagonist at the BRCA-1 gene with IC50 approaching ~0.4 mM (53), and a potent aromatase inhibitor. It shares structural similarity with GEN and the flavonol galangin (3,5,7-trihydroxyflavone, IC50 ~0.2 mM), which blocks proliferation of ERa-negative breast cancer cells overexpressing AhR. Finally, we reported that treatment of UACC-3199 cells with GEN and aNF activated preferentially CYP1A1 with only modest (aNF) or no (GEN) effects on CYP1B1. This selective activation of CYP1A1 may have therapeutic relevance since increased CYP1A1 expression associates with reduced basal AhR activity and increased apoptosis. Furthermore, reduction of 4-hydroxylation of E2 by aNF, a reaction catalyzed by CYP1B1, has been shown to reduce the production of the highly carcinogenic metabolite 4-hydroxy-E2 and mammary tumorigenesis. Overall, results of this study suggested preventative effects for GEN and EGCG against proliferation and AhR-mediated BRCA-1 CpG methylation in ERa-positive breast cancer cells. We also presented evidence that GEN and aNF, a prototype flavone and AhR antagonist, may hold promise for reactivation of BRCA-1 in ERa-negative sporadic breast tumors with constitutively activate AhR.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Romagnolo DF, Selmin OI. Mediterranean Diet and Lifestyle in a Modern World Context. In Mediterranean Diet: Impact on Health and Disease. Humana Press/Springer, 2016, pp15-26
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Selmin OI, Romagnolo APG, Romagnolo DF. Mediterranean Diet and Neurodegenerative Diseases. In Mediterranean Diet: Impact on Health and Disease. Humana Press/Springer. 2016, pp153-164.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Donovan MG, Selmin OI, Doetschman TC, Romagnolo DF. Mediterranean Diet, Inflammatory Bowel Diseases and Colon Cancer. In Mediterranean Diet: Impact on Health and Disease. Humana Press/Springer. 2016, pp181-216.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Selmin OI, Romagnolo DF. Building the Mediterranean Pyramid-PART A: Mediterranean Recipes. In Mediterranean Diet: Impact on Health and Disease. Humana Press/Springer. 2016, pp261-273.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Romagnolo DF, Jackson K, Sparks P, Selmin OI. Building the Mediterranean Pyramid: Part BBalancing the Plate. In Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease. Human Press/Springer. 2016, pp275288.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Romagnolo DF, Selmin OI. Appendix: 2015 Dietary Guidelines for Americans. Humana Press/Spinger. 2016, pp289-311.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Romagnolo, D. (2016). Epigenetics of endocrine tumors: modifying role of environmental and bioactive food compounds. Mol Nutr Food Res. 2016, 60:1310-29.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Target audiences include nutrition scientists, undergraduate, graduate and postdoctoral fellows in training; nutrition clinicians; and nutrition practicing professionals; food industry; and representatives of state and federal funding agencies;and the general public at large. Changes/Problems:No major changes to the research strategy were necessary. What opportunities for training and professional development has the project provided?The project has provided opportunities for training of undergraduate students, graduate students and professional development of scientists. How have the results been disseminated to communities of interest?Results were presented at various conferences including: 2015 Experimental Biology Meetings, ASN. Boston, MA. Adam Lyon, Ornella Selmin, Changming Fang, Jeffrey W. Smith, Tom Doetschman, Patricia Thompson, Jesse Martinez, Peter Lance, Donato Romagnolo. Epigenetic Regulation of the FXR by High Fat Diet and APC in Colon Cells. Research Frontiers in Nutritional Sciences Conference. The University of Arizona, Tucson, AZ. January 28-30, 2015.Jenna Radomski, Ornella Selmin, Donato Romagnolo. Mediterranean Diet and Health Study Abroad Program. What do you plan to do during the next reporting period to accomplish the goals?We will continue our investigations of the effects of AHR activation on regulation of DNA methylation using global methylation assays and in response to dietary antagonists of the AhR; Continue studies of high fat diet and regulation of bile acid homeostasis. test the effects of isoflavones (i.e. genistein) on BRCA-1 methylation in mammary epithelial cells.
Impacts
What was accomplished under these goals?
(1) Determine the mechanisms by which dietary bioactive compounds protect against human diseases. Background: Only 5-10% of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95% of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy. Methods: We evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague-Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples. Results: Mammary tumors induced with DMBA had reduced BRCA-1 and ERa expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. TNBC exhibited increased basal AhR and BRCA-1 promoter CpG methylation compared to LUM-A, LUM-B, and HER-2-positive breast tumors. Conclusions: Constitutive AhR expression coupled to BRCA-1 promoter CpG hypermethylation may be predictive markers of ERa-negative breast tumor development. (2) Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention. Background: The farnesoid X receptor (FXR) regulates bile acids (BA) metabolism and possesses tumor suppressor functions. The expression of FXR is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. Objective: The objective of this study was to investigate the influence of APC inactivation on regulation of FXR expression in colonic mucosal cells. We hypothesized that inactivation of APC epigenetically represses NR1H4 (FXR gene name) expression through increased CpG methylation. Methods: Normal proximal colonic mucosa, and normal-appearing adjacent colonic mucosa and colon tumors were collected respectively from wild-type C57BL/6J and Apc deficient (ApcMin/+) male mice. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time PCR. In both normal, and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC (siAPC), and HT-29 cells carrying inactivated APC.Results: In ApcMin/ +mice, constitutive CpG methylation of the Fxra3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp, and increased Cox-2, expression in apparently normal adjacent mucosa and colon tumors. The knock-down of APC in HCT-116 cells increased c-MYC, and lowered (~50%) FXR expression, which was further reduced (~80%) by DCA. In human HCT-116, but not HT-29, colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. Conclusions: We conclude that in mouse colonic mucosa and human colon cells loss of APC function favors silencing of FXR expression through CpG hypermethylation leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer. (3) Discover and characterize novel bioactive dietary compounds that have beneficial or adverse effects on human health. Background: understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy. Methods: We tested in human estrogen receptor (ER)a-negative sporadic UACC-3199 and ERa-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist a-naphthoflavone (aNF) modulated BRCA-1 and ERa expression. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with aNF rescued BRCA-1 and ERa, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, aNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERa. Conclusions: Regimens based on selected AhR modulators (SAhRMs) may be useful for therapy against ERa-negative tumors, and possibly, TNBC with increased AhR and hypermethylated BRCA-1 gene.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Romagnolo AP, Romagnolo DF, Selmin OI. BRCA1 as target for breast cancer prevention and therapy. Anticancer Agents Med Chem. 2014;15(1):4-14.
Selmin OI, Fang C, Lyon AM, Doetschman TC, Thompson PA, Martinez JD, Smith JW, Lance PM, Romagnolo DF. Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells. J Nutr. 2015 Nov 25. pii: jn216580.
Romagnolo DF, Papoutsis AJ, Laukaitis C, Selmin OI. Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ER-negative breast tumorigenesis. BMC Cancer. 2015 Dec 29;15(1):1026.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
FXR in APC deficiency and characterization of FXR transgenic mice. Adam Lyon, A Thesis submitted to the Faculty of the Department of Nutritional Sciences in partial fulfillment of the requirements for degree of Master of Science.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Adam Lyon, Ornella Selmin, Changming Fang, Jeffrey W. Smith, Tom Doetschman, Patricia Thompson, Jesse Martinez, Peter Lance, Donato Romagnolo. Epigenetic Regulation of the FXR by High Fat Diet and APC in Colon Cells. 2015 Experimental Biology Meetings, ASN. Boston, MA.
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Romagnolo DF, Selmin OI. Epigenetics of Endocrine Tumors in Women and Dietary Prevention. In: Preventive Nutrition, Fifth Edition, Eds. Humana Press/Springer, 2015.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target audiences include nutrition scientists, undergraduate, graduate and postdoctoral fellows in training; nutrition clinicians; and nutrition practicing professionals; food industry; and representatives of state and federal funding agencies;and the general public at large. Changes/Problems: No major changes were made to the work plan. What opportunities for training and professional development has the project provided? The project has provided opportunities for training of undergraduate students, graduate students and professional development of scientists. How have the results been disseminated to communities of interest? Results have been disseminated through presentations at national meetings (Faseb 2014, San Diego, CA); State and Regional Conferences (Research Frontiers in Nutritional Sciences); and Departmental Seminars at The University of Arizona. What do you plan to do during the next reporting period to accomplish the goals? We will continue our investigations of the effects of AHR activation on regulation of DNA methylation using global methylation assays and in response to dietary antagonists of the AhR; Continue studies of high fat diet and regulation of bile acid homeostasis.
Impacts
What was accomplished under these goals?
1. Determine the mechanisms by which dietary bioactive compounds protect against human diseases. Post-translational modifications of histones, alterations in the recruitment and functions of non-histone proteins, DNA methylation, and changes in expression of non-coding RNAs contribute to current models of epigenetic regulation. Nuclear receptors (NRs) are a group of transcription factors that through ligand-binding act as sensors to changes in nutritional, environmental, developmental, pathophysiological, and endocrine conditions, and drive adaptive responses via gene regulation. 2. Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention. One mechanism through which NRs direct gene expression is the assembly of transcription complexes with cofactors and coregulators that possess chromatin modifying properties. Chromatin modifications can be transient or become part of the cellular "memory" and contribute to genomic imprinting. 3. Discover and characterize novel bioactive dietary compounds that have beneficial or adverse effects on human health. Because many food components bind to NRs, they can ultimately influence transcription of genes associated with biological processes such as inflammation, proliferation, apoptosis, hormonal response, and alter the susceptibility to chronic diseases (e.g. cancer, diabetes, obesity). Our work highlights how NRs influence epigenetic regulation, and the relevance of dietary compound-NR interactions in human nutrition, and for disease prevention and treatment. Identifying gene targets of unliganded and bound NRs may assist in the development of epigenetic maps for food components and dietary patterns. Progress in these areas may lead to the formulation of disease-prevention models based on epigenetic control by individual or associations of food ligands of NRs.
Publications
- Type:
Books
Status:
Awaiting Publication
Year Published:
2015
Citation:
Romagnolo Df, and Selmin OI. Co-Editors. Mediterranean Diet: Impact on Health and Disease. Humana Press/Springer (2014, In Press, book approved for publication).
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Romagnolo DF, Zempleni J, Selmin OI. Nuclear receptors as epigenetic modifiers of gene expression in chronic diseases. Adv Nutr 2014. May 1, 1-13.
Romagnolo AP, Romagnolo DF, Selmin OI. BRCA1 as target for breast cancer prevention and therapy. Anticancer Agents Med Chem. 2014;15(1):4-14.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Romagnolo DF, Selmin OI. Epigenetics of BRCA-1-related breast tumorigenesis and dietary prevention. In The Role of Nutrition and Metabolism on Epigenetic Regulation Taylor & Francis Group, CRC Press. E. Ho and F Domann, Eds. 2014.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Selmin OI, Papoutsis JA, and Romagnolo DF. Postpubertal exposure to the AhR agonist and carcinogen DMBA (7,12-dimethylbenz[?]anthracene) induces mammary tumors with hypermethylated BRCA-1 promoter and constitutive AhR expression in C57BL/6 mice. Breast Cancer Res. 2014 (submitted).
Selmin OI, Martinez J. Lance P, Smith J, Romagnolo DF. Epigenetic repression of FXR in colonic mucosa: modifying roles of APC and bile acids. Mol Carcinogenesis, 2014 (submitted).
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2015
Citation:
Romagnolo DF and Selmin OI. Opportunities and Challenges for Adoption of the Mediterranean Diet. In: Mediterranean Diet: Impact on Health and Disease. Humana Press/Springer Pubs (In press).
Romagnolo DF and Selmin OI. Epigenetics of Endocrine Tumors in Women and Dietary Prevention. In: Preventive Nutrition, Fifth Edition, Eds. Humana Press/Springer (In press).
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Audiences include nutrition scientists, clinicians, students and postdocs in training, nutrition professionals involved in extension, and the public at large. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Findings were disseminated through presentations at local, national, and international meetings, and seminars. This work contributed to supporting a doctoral dissertation program (Dr. Andreas Papoutsis) in Nutritional Sciences and Cancer Biology at The University of Arizona. How have the results been disseminated to communities of interest? Donato F. Romagnolo. Epigenetics of Breast Cancer. MARC lecture. The University of Arizona. March 18, 2013. Donato F. Romagnolo. Bioactive food compounds and epigenetic mechanisms in breast cancer. 2013 Diet and Optimum Health Conference. Linus Pauling Institute and Oxygen Club of California, Oregon State University. May 17, 2013. What do you plan to do during the next reporting period to accomplish the goals? Data generated have raised important questions with significant impact for the development of molecular signatures and therapies against ERa-negative breast tumors. For the next reporting period we plan to address the following questions: · Does the AhR directly mediate the epigenetic changes at the BRCA-1 gene and other factors that contribute to the triple negative breast cancer (TNBC) phenotype? · Do interactions between AhR and BRCA-1 genotype influence the risk of TNBC? Sporadic breast cancers that have CpG methylated BRCA-1 promoter share phenotypic characteristics (BRCAness) with hereditary BRCA-1 mutation tumors, i.e. they tend to cluster with TNBC. Therefore, answering these questions may provide the basis for the development of therapeutic strategies for women who carry mutated or inactivated BRCA-1, and develop TNBC, for which prospects for prevention and treatment remain unclear.
Impacts
What was accomplished under these goals?
Specific objectives of this project were: 1) To investigate the mechanisms of inactivation of the BRCA-1 gene by ligands of the aromatic hydrocarbon receptor (AhR). The AhR is a nuclear receptor that acts as a sensor to many compounds including those found in diet and pollution, fatty acid metabolites, and photoproducts in the skin; 2) To examine the preventative effects of dietary antagonists of the AhR on BRCA-1 expression and mammary development. The work conducted under the support of thisproject led to the following outcomes: We found that the gestational activation of the AhR increased CpG methylation at the BRCA-1 promoter and reduced BRCA-1 protein expression in mammary tissue of female rat offspring. Increased CpG methylation represents an epigenetic silencing event accompanied by reduced expression of BRCA-1. These changes were overridden by pretreatment with the AhR antagonist resveratrol, which is found in grapes, blueberries, and peanuts. Also, we gathered evidence that the in utero activation of the AhR impaired mammary differentiation in offspring, as indicated by the increased number of terminal ducts and terminal end buds, and the accumulation of the proliferation markers cyclin D1 and cyclin-dependent kinase-4 (cdk4) in mammary tissue of offspring. These changes are recognized predisposing events of mammary tumorigenesis. The conclusion from these studies is that maternal exposure to AhR agonists is a risk factor for the development of breast cancer in the female offspring and that the dietary AhR antagonists can offer protection. We obtained evidence that mammary tumors induced in postpuberty with an AhR-agonist [DMBA (7,12-dimethylbenz[α]anthracene)] exhibited increased BRCA-1 promoter CpG methylation, and reduced BRCA-1 and ERa protein expression. In addition, we observed that apparently normal mammary tissue isolated from DMBA-treated animals harbored increased BRCA-1 promoter CpG methylation compared to control tissue. The conclusion from these studies is that methylation changes at the BRCA-1 gene may constitute a marker for the early diagnosis of ERa-negative breast tumors induced by AhR agonists, and offer opportunities for targeted prevention with dietary AhR and ER antagonists.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Papoutsis AJ, Selmin OI, Borg JL, and Romagnolo DF. 2013. Gestational Exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin Induces BRCA-1 Promoter Hypermethylation and Reduces BRCA-1 Expression in Mammary Tissue of Rat Offspring: Preventive Effects of Resveratrol. Mol. Carcinog. 2013 Oct 17. doi: 10.1002/mc.22095.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Romagnolo DF and Selmin OI. 2013 Herbal Supplements in the Prevention and Treatment of Cancer. Handbook of Nutrition and Food, Third Ed. Taylor and Francis Pubs.
- Type:
Book Chapters
Status:
Accepted
Year Published:
2013
Citation:
Romagnolo DF. 2014. Bioactive food compounds and epigenetic mechanisms in breast cancer. In The Role of Nutrition and Metabolism on Epigenetic Regulation Taylor & Francis Group, CRC Press. E. Ho and F Domann, Eds. (In press)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Selmin OI and Romagnolo DF. 2014 Epigenetic Regulation by Nuclear Receptors and Nutritional Targeting of Chronic Diseases. Adv. Nutr.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Selmin OI, Papoutsis JA, and Romagnolo DF. Postpubertal exposure to the AhR agonist and carcinogen DMBA ((7,12-dimethylbenz[?]anthracene) induces mammary tumors with hypermethylated BRCA-1 promoter and constitutive AhR expression in C57BL/6 mice. Breast Cancer Res.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Activities: 1. Does exposure to ligands of the aromatic hydrocarbon receptor influence the risk of disease Epigenetic mechanisms contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorobenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XRE=GCGTG) and interactions with transcription cofactors. We conducted experiments to investigate if XREs present in the proximal BRCA-1 promoter the expression of endogenous AhR mediated silencing of BRCA-1 expression by TCDD. These activities have been used to mentor Ph.D. students (Andreas Papoutsis) and train staff (Jamie Borg); data were presented at the 2012 Experimental Biology Meetings in the form of a symposia in San Diego, CA. Dissemination of this information occurred through seminars presented by Andreas Papoutsis on ther Campus of the University of Arizona; class presentations by Dr. Romagnolo, specifically to students enrolled in Nutritional Biology NSC408 and Metabolic Integration NSC602; and publication in scientific journals. Products include one Ph.D. student (Dr. Papoutsis)graduated in nutritional sciences and development of laboratory skills. 2. Are dietary flavonoids protective against cancer The objective of this work was to review data from epidemiological and preclinical studies addressing the potential benefits of diets based on flavonoids for cancer prevention. Flavonoids are subdivided into subclasses including flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones. Epidemiological studies suggest dietary intake of flavonoids may reduce the risk of tumors of the breast, colon, lung, prostate, and pancreas. However, some studies have reported inconclusive or even harmful associations. 3. Are proteomic tools useful for studies of cancer prevention through dietKnowledge gaps persist about the efficacy of cancer prevention strategies based on dietary food components. Adaptations to nutrient supply are executed through tuning of multiple protein networks that include transcription factors, histones, modifying enzymes, translation factors, membrane and nuclear receptors, and secreted proteins. However, the simultaneous quantitative and qualitative measurement of all proteins that regulate cancer processes is not practical using traditional protein methodologies. PARTICIPANTS: Individuals from the University of Arizona included Dr. Donato Romagnolo, principal investigator; Dr. Ornella Selmin, collaborator; Andreas Papoutsis, who received his Ph.D. under supersivion of Dr. Romagnolo; and Jamie Borg. Collaborators were: Dr. Dashwood R., from Oregon State University; Dr. Stover PJ, from Cornell University; Dr. Waterland RA, from Baylor College of Medicine; Dr Ziegler TR, from Emory University; and Dr. DeRoos B, from the University of Aberdeen, UK. TARGET AUDIENCES: Target audiences for this work included undergraduate and graduate students attending the University of Arizona; principal investigators and scientists in training on the Campus of the University of Arizona; nutrition and clinical scientists in the US, and the general public at large in the State of Arizona and the US. PROJECT MODIFICATIONS: There were no major changes in approach.
Impacts
Change in knowledge: ACTIVITY 1: We found that in estrogen receptor alpha (ERalpha)-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17beta estradiol (E2)-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR, DNA methyl transferases (DNMT)1, DNMT3a, and DNMT3b; methyl binding protein (MBD)2; and tri-methylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses (1 micromol/L) the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation, and the recruitment of the AhR, MBD2, H3K9me3, and DNMTs (1, 3a, and 3b). These observations provide mechanistic evidence for AhR-agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of preventive actions based on AhR antagonists. ACTIVITY 2: A major challenge in the interpretation of epidemiological studies is that most of the data originate from case-control studies and retrospective acquisition of flavonoid intake. Differences in agricultural, socio-demographics, and lifestyle factors contribute to the heterogeneity in the intake of flavonoids among populations residing in the U.S., Europe, and Asia. Dose and timing of exposure may influence the anticancer response to flavonoid-rich diets. A limited number of intervention trials of flavonoids have documented cancer preventative effects. Proposed anticancer mechanisms for flavonoids are inhibition of proliferation, inflammation, invasion, metastasis, and activation of apoptosis. Prospective studies with larger sample sizes are needed to develop biomarkers of flavonoid intake and effect. Mechanistic studies are needed to ascertain how flavonoid-rich diets influence gene regulation for cancer prevention. ACTIVITY 3. Proteomics offers an attractive opportunity to fill a knowledge gap and unravel the effects of dietary components on protein networks that impinge on cancer. Recent advances in MS technologies suggest that studies in nutrition and cancer prevention may benefit from the adoption of proteomic tools to elucidate the impact on biological processes that govern the transition from normal to malignant phenotype; to identify protein changes that determine both positive and negative responses to food components; to assess how protein networks mediate dose-, time-, and tissue-dependent responses to food components; and, finally, for predicting responders and nonresponders.
Publications
- Romagnolo DF, Dashwood R, Stover PJ, Waterland RA, Ziegler TR. Nutritional regulation of epigenetic changes. Adv Nutr. 2012 Sep 1;3(5):749-50. doi: 10.3945/an.112.002675. PubMed PMID: 22983864.
- 2: Romagnolo DF, Selmin OI. Flavonoids and cancer prevention: a review of the evidence. J Nutr Gerontol Geriatr. 2012;31(3):206-38. doi: 10.1080/21551197.2012.702534. PubMed PMID: 22888839.
- 3: Romagnolo DF, Davis CD, Milner JA. Phytoalexins in cancer prevention. Front Biosci. 2012 Jun 1;17:2035-58. Review. PubMed PMID: 22652763.
- 4: Romagnolo DF, Milner JA. Opportunities and challenges for nutritional proteomics in cancer prevention. J Nutr. 2012 Jul;142(7):1360S-9S. Epub 2012 May 30. PubMed PMID: 22649262; PubMed Central PMCID: PMC3374671.
- 5: de Roos B, Romagnolo DF. Proteomic approaches to predict bioavailability of fatty acids and their influence on cancer and chronic disease prevention. J Nutr. 2012 Jul;142(7):1370S-6S. Epub 2012 May 30. Review. PubMed PMID: 22649259; PubMed Central PMCID: PMC3374672.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Outputs included: 1) Presentation of lectures for Nutritional Biology NSC408 on impact of environmental exposure and risk of cancer; 2)Mentoring of a graduate student (Andreas Papoutsis) and development of a project targeted to investigating the effects of exposure to ligands of the aromatic hydrocarbon (AhR) on breast cancer risk; 3) Development of a Symposium on Epigenetics for the American Society of Nutrition; 4) Development of a collaboration with the Laboratory of Proteomics, NCI-SAIC, Frederick; 5)Development of DNA methylation techniques to assess epigenetic regulation of tumor suppressor genes; 6) Dissemination of information through organization of the Research Frontiers in Nutritional Sciences Conference; 7) Presentation of Seminars at the Arizona Cancer Center and lectures to Cancer Biology Program at The University of Arizona PARTICIPANTS: Individuals: Papoutsis AJ (graduate student), Borg JL (Technician), Selmin OI (Associate Professor), Romagnolo DF (Professor), Department of Nutritional Sciences, The University of Arizona, Tucson, AZ. Palbykin B (Undergraduate student, Molecular Biology), Caldwell PT (graduate student, Veterinary Science and Microbiology), Rowles J (undergraduate student, Molecular Biology. The University of Arizona, Tucson, AZ. Collaborators: JA Milner,National Cancer Institute, NIH. The current project offered the opportunity for training of Andreas Papoutsis, Ph.D. candidate in Nutritional Sciences and professional development of Jamie Borg, technician, in various laboratory techniques. TARGET AUDIENCES: Target audience includes basic nutritionists and dietitians, medical practitioners, cancer biologists, undergraduate and graduate students and individuals involved in nutrition education/extension. The change in knowledge provided by this project is that it documents the mechanisms of action of certain food components in the prevention of epigenetic alterations associated with breast cancer. The long-term impact of the findings of this progress report is that they may offer the basis for the development of new prevention strategies. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have investigated the mechanisms responsible for the reduced expression of BRCA-1 in breast tissue. Our data suggest that in MCF-7 cells, the activation of the AhR with TCDD increases the association with the BRCA-1 promoter of the histone deacetylase-1 (HDAC-1) while reducing the recruitment of ERalpha, p300, SRC-1, and acetylated H4 (AcH4). Loss of AcH4 and gain of HDAC activity are typically associated with transcriptional repression. Conversely, transfection into MCF-7 cells of siRNA for the AhR restored BRCA-1 protein expression. The increased occupancy of the AhR at the BRCA-1 promoter was paralleled by enhanced association of DNA methyl transferases (DNMT-1, 3a, 3b), methyl binding protein 2 (MBD2), and hypermethylation of the CpG island spanning the transcriptional start site of BRCA-1 exon-1a. Reduced BRCA-1 expression has been reported in sporadic breast tumors that overexpress DNMT3b. Also, we found that increased MBD2 binding has been reported in methylated promoters including p16 and 14-3-3sigma. Conversely, the AhR antagonist resveratrol antagonized BRCA-1 promoter hypermethylation induced by TCDD. These cell culture data support a possible role for the AhR in silencing of BRCA-1 through promoter hypermethylation and the preventative role of a dietary AhR antagonist at physiologically relevant doses.
Publications
- Tricholoroethylene induces methylation of the Serca2 promoter in H9c2 cells and embryonic heart. Palbykin B, Borg J, Caldwell PT, Rowles J, Papoutsis AJ, Romagnolo DF, Selmin OI. Cardiovascular Toxicology. 2011 Sep;11(3):204-14.
- Phytoalexins in Nutrition and Cancer Prevention Romagnolo DF, JA Milner. 2011. Frontiers in Biosciences. Accepted for publication (In Press.)
- Opportunitites and Challenges for Nutritional Proteomics in Cancer Prevention. Romagnolo DF, JA Milner, 2011. Journal of Nutrition. Accepted for Publication, (In Press).
- BRCA-1 promoter hypermethylation and silencing induced by the aromatic hydrocarbon receptor-ligand TCDD are prevented by resveratrol in MCF-7 cells. Papoutsis AJ, Borg JL, Selmin OI, Romagnolo DF. Journal of Nutritional Biochemistry. 2011 Dec 22.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Objective 1. Investigate the mechanisms of epigenetic repression of BRCA-1 by the activated AhR and prevention by dietary AhR antagonists. The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the aromatic hydrocarbon receptor (AhR), which contribute to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen receptor- alpha (ER alpha )-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. Objective 2. This was an exploratory study developed to investigate the opportunities for targeting of cyclooxygenase-2 (COX-2) expression with bioactive food components for the prevention of inflammation and colon cancer. Factors related to diet and life style have been identified as primary determinants in about 80% of colorectal cancers. Non-steroidal anti-inflammatory drugs (NSAID) and selective (COX-2) inhibitors (COXIB) reduce the relative risk of colon cancer. To overcome systemic COX inhibition associated with NSAID and COXIB, there is a growing interest in developing alternative colon cancer prevention strategies using diet-based approaches that target COX-2. The transition from aberrant crypt foci (ACF) to colon cancer is a multiyear process providing opportunities for nutritional targeting of genes influencing the course of this disease process at early stages of development. The activation of the proinflammatory gene COX-2 and PG production in the colonic mucosa are recognized risk factors in colon cancer. Many natural food components may impact colon cancer risk by interfering with ligand-activated receptors, signal transduction pathways, and transcription factors involved in stimulation of COX-2 expression. In a systematic review of published studies, we highlighted key upstream features of signaling pathways and transcriptional control of the COX-2 gene and discussed opportunities for dietary modulation of COX-2 expression in gastro-intestinal cancers with special emphasis on prevention of colorectal tumors. This information has been disseminated through lectures offered to undergraduate and graduate classes (NSC 408 and NSC 602), seminar lectures to graduate students in training (Seminar series, Department of Nutritional Sciences), and conferences (Nutritional Sciences Conference, The University of Arizona) PARTICIPANTS: Individuals who participated included: Romagnolo DF, Papoutsis AJ, Selmin O, Sarah Lamore, Jamie Borg, and and George Wondrak, The University of Arizona, Tucson. Opportunities for training included the development of a research project for Andreas Papoutsis, who is a Ph.D. student in Nutritional Sciences and professional development of Jamie Borg, Research Technician. TARGET AUDIENCES: Target audiences include graduate students in training, basic nutrition scientists, nutrition professionals, and the general public at large. Information gained through this project may increase the awarness of the scientific community, health practitioners and the general public about the causes related to inflammation and cancer and how diet may prevent these chronic conditions and diseases. PROJECT MODIFICATIONS: None
Impacts
We found that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17 beta -estradiol (E2)-dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ER alpha, acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists. Review of the experimental evidence suggests that dietary strategies based on specific or cocktails of bioactive food components as well nutritional-pharmacological combinations targeted to regulation of COX-2 expression and activity may prove useful in the prevention of colon cancer. An integrated approach may offer the advantage of combined higher efficacies. Future studies should investigate the efficacy of combinations of bioactive food compounds on epigenetic regulation of the COX-2 gene and characterize potential synergies and amplification effects resulting from the concomitant use of bioactive food components and COX-2 inhibitors.
Publications
- Papoutsis AJ, Lamore SD, Wondrak GT, Selmin OI, Romagnolo DF. Resveratrol prevents epigenetic silencing of BRCA-1 by the aromatic hydrocarbon receptor in human breast cancer cells. J Nutr. 2010 Sep;140(9):1607-14. Epub 2010 Jul 14.
- Romagnolo DF, Papoutsis AJ, Selmin O.Nutritional targeting of cyclooxygenase-2 for colon cancer prevention. Inflamm Allergy Drug Targets. 2010 Jul 1;9(3):181-91.
- Milner J.A., and Romagnolo D.F. Nutrigenomics and Cancer Biology. In: Bioactive Food Components and Cancer, Humana Press/Springer. Eds. Milner and Romagnolo (2010).
- Romagnolo D.F., Degner SC, and Selmin, O. Aryl Hydrocarbon Receptor-mediated Carcinogenesis and Modulation by Dietary Xenobiotic and Natural Ligands, In: Bioactive Food Components and Cancer, Humana Press/Springer. Eds. Milner and Romagnolo.(2010).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Results of this project have been disseminated and shared through presentations at local, national, and international meetings including presentations at the Seminar series of the Nutritional Sciences Department at The University of Arizona, seminars presented at the Arizona Cancer Center, presentations of poster material at the Annual meetings of the American Institute for Cancer Research and Annual meetings of the American Association of Nutrition; presentations at the 2009 Research Frontiers in Nutritional Sciences Conference organized by the Department of Nutritional Sciences, and publication of original research articles, and review papers.Individuals who contributed to this results and participated in the dissemination include graduate students (Stephanie Degner, Andreas papoutsis), scientists (Ornella Selmin) and colleagues (Geor Wondrak). Target audiences included the scientific community at the University of Arizona, colleagues at US and international Universities, research foundations, and the public at large through publication of scientific articles and book chapters. PARTICIPANTS: Individuals who contributed to this work included graduate students (Stephanie Degner, Andreas Papoutsis), collaborators in the Nutritional Sciences Department (Ornella Selmin, Berggren M, Sittadjody S, Song Z, Samira JL, Burd R, Meuillet EJ ), Department of Pharmacology and Toxicology (Georg Wondrak), University of Maryland and American Institute for Cancer Research (Ivana Vucenik), and the National Cancer Institute (J. Milner). TARGET AUDIENCES: Target audiences included the scientific community at the University of Arizona, colleagues at US and international Universities, health practitioners, research foundations, and the public at large through publication of articles and book chapters. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objevtive 1. The project contributed to improve our understanding how dietary factors may regulate the expression of tumor suppressor genes and how these mechanisms may influence the risk of breast cancer and the development of prophylactic dietary strategies. The central hypothesis of this project is that natural and environmental ligands of the aromatic hydrocarbon receptor (AhR) lead to the recruitment of AhR to the promoter region of BRCA-1 causing chromatin modifications that culminate with silencing of the BRCA-1 gene. Potential impacts of this project are the development of nutritional strategies that protect against the development of sporadic breast cancer and other BRCA-1 related tumors. Results of the BRCA-1 project indicate that the tumor suppressor gene BRCA-1 can be repressed by ligands of the AhR, which include environmental polycyclic aromatic hydrocarbons and dioxins, and many natural bioactive compounds found in diet. Many AhR-ligands are found in vegetables, fruits, tea, and herbs and their specific metabolites may act as agonists or antagonists of the AhR depending on molecular structure, concentration, tissue- and cell-type, target gene, and time of exposure. The cumulative exposure to agents that act as AhR-ligands may lead to epigenetic silencing of tumor suppressor genes, such as BRCA-1, thus increasing the susceptibility to sporadic breast cancer in the absence of mutations in the BRCA-1 gene. The exposure to synthetic or natural analogues of the AhR may, through modulation of DNA methylation patterns, lead to heritable changes in the expression of target genes and to long-term effects on human health. However, very little is known about how individual AhR-ligands influence the epigenetic process, and the impact of levels of intake and timing of exposure on tissue specificity. Specific contributions include studies of how the grape compound resveratrol regulates BRCA-1 expression through modifications of chromatin organization at the BRCA-1 gene. These results provide the fundamental observations to develop preventative strategies based on antagonists of the AhR. Objective 2. Epidemiological and clinical data suggest that selenium may prevent prostate cancer; however, the cellular effects of selenium in malignant prostate cells are not well understood. We previously reported that the activity of the tumor suppressor PTEN is modulated by thioredoxin (Trx) in a RedOx-dependent manner. In this study, we demonstrated that the activity of Trx reductase (TR) is increased by sevenfold in the human prostate cancer cell line, DU-145, after 5 days of sodium selenite (Se) treatment. The treatment of DU-145 cells with increasing concentrations of Se induced an increase in PTEN lipid phosphatase activity by twofold, which correlated with a decrease in phospho-ser(473)-Akt, and an increase in phospho-Ser(370)-PTEN levels. Se also increased casein kinase-2 (CK2) activity; and the use of apigenin, an inhibitor of CK2, revealed that the regulation of the tumor suppressor PTEN by Se may be achieved via both the Trx-TR system and the RedOx control of the kinase involved in the regulation of PTEN activity.
Publications
- Degner SC, Papoutsis AJ, Selmin O, Romagnolo DF. Targeting of aryl hydrocarbon receptor-mediated activation of cyclooxygenase-2 expression by the indole-3-carbinol metabolite 3,3'-diindolylmethane in breast cancer cells. J Nutr. 2009 Jan;139(1):26-32.
- Papoutsis AJ, Lamore SD, Wondrak GT, Selmin IO, Romagnolo DF. Epigenetic Regulation of BRCA-1 by AhR Agonists and Preventative Effects of Resveratrol in Breast Cancer Cells. 2009 (submitted).
- Romagnolo D.F., and I. Vucenik. Cancers of the Female Reproductive System. In: Preventive Nutrition, Fourth Edition, Eds. Humana Press/Springer, 2009).
- Berggren M, Sittadjody S, Song Z, Samira JL, Burd R, Meuillet EJ. Sodium selenite increases the activity of the tumor suppressor protein, PTEN, in DU-145 prostate cancer cells. Nutr Cancer. 2009;61(3):322-31. PubMed PMID: 19373605.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Dissemination of results was through presentations to national and international meeetings, Departmental Seminars, and University of Arizona activities: Degner S.C., Scheckel K.A., and Romagnolo D.F. Federation of the American Society of Experimental Biology/American Society of Nutrition, Annual Meetings, San Diego, CA, 2008. Degner S. C., Papoutsis, A.J., Scheckel, K.A., Selmin O., and Romagnolo D.F. Targeting of aryl hydrocarbon receptor-regulated COX-2 expression in breast cancer cells. 2008 Meetings of the American Institute for Cancer Research (AICR), Washington, D.C., 2008. D.F. Romagnolo. Linking epidemiological evidence with mechanistic evidence in cancer prevention. Department of Nutritional Sciences Seminar Series. Dec 9, 2008. PARTICIPANTS: Individuals who contributed to this project include graduate students (Stephanie Degner, Andreas Papoutsis), undergraduate students enrolled in the Honors College (Kristen Sheckel), and colleagues from the University of Arizona (Dr. Ornella Selmin). This work provided the opportunity for the development of a Ph.D. Dissertation by Stephanie Degner, scientific projects for the Honors College Undergraduate Research Program by Kristen Sheckel, and publications and presentations at local, national, and international scientific meetings. TARGET AUDIENCES: Target audiences included the scientific community at the University of Arizona, colleagues at US and international Universities, research foundations, and the public at large through publication of articles and book chapters. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The long-term objective of this project is to examine how changes in the expression of tumor suppressor and pro-inflammatory genes influence the risk of breast cancer and develop prophylactic dietary strategies. The central hypothesis of this project is that natural and environmental ligands of the aromatic hydrocarbon receptor (AhR) lead to the recruitment of AhR to the promoter region of BRCA-1 or COX-2 causing chromatin modifications that culminate with silencing of the tumor suppressor gene BRCA-1, or activation of the proinflammatory gene, COX-2. Potential impacts of this project are the development of nutritional strategies that protect against the development of sporadic breast cancer. Results of the BRCA-1 project indicate that the tumor suppressor gene BRCA-1 can be repressed by ligands of the AhR, which include environmental polycyclic aromatic hydrocarbons and dioxins, and many natural bioactive compounds found in diet. Many AhR-ligands are found in vegetables, fruits, tea, and herbs and their specific metabolites may act as agonists or antagonists of the AhR depending on molecular structure, concentration, tissue- and cell-type, target gene, and time of exposure. The cumulative exposure to agents that act as AhR-ligands may lead to epigenetic silencing of tumor suppressor genes, such as BRCA-1, thus increasing the susceptibility to sporadic breast cancer in the absence of mutations in the BRCA-1 gene. The exposure to synthetic or natural analogues of the AhR may, through modulation of DNA methylation patterns, lead to heritable changes in the expression of target genes and to long-term effects on human health. However, very little is known about how individual AhR-ligands influence the epigenetic process, and the impact of levels of intake and timing of exposure on tissue specificity. Conversely, AhR-ligands can activate the expression of COX-2 thus increasing the risk of inflammation and cancer. Therefore, the concomitant epigenetic repression of tumor suppressor genes and activation of proinflammatory genes by AhR-ligands could be prevented through dietary interventions.
Publications
- Sheckel K.A., Degner S.C., and Romagnolo D.F. 2008. Rosmarinic Acid Antagonizes Activator Protein-1-Dependent Activation of Cyclooxygenase-2 Expression in Human Cancer and Non-Malignant Cell Lines. J. Nutr. 138:2098-2105.
- Degner S.C., and Romagnolo D.F. Phytochemicals and Anti-Cancer Mechanisms. In: Botanical medicine in Clinical Practice, Ed. Ronald Watson. CAB International, Oxon, UK, Chapter 40, pg. 386-399, 2008.
- Degner S.C., Papoutsis A.J. and Romagnolo D.F. Health Benefits of Traditional Mediterranean Herbs and Spices. In: CAM Botanical Therapies to Promote Health in the Aged, Ed. Ronald Watson. Chapter 26, pg. 541-562, 2008.
- Romagnolo D.F, Papoutsis A.J. S.C. Degner, and O. Selmin. Linoleic Acids and Cancer Cell Functions. In: Fatty Acids in Health Promotion and Disease Causation. AOCS. Ed. Ronald Watson, 2008.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The work reported is an outcome of the undergraduate research project of Kristen Scheckel, undergraduate student enrolled in The Undergraduate Research Program of The Honors College at The University of Arizona. This work was presented at the @1st Annual Undergraduate Research Forum. Data are also scheduled to be presented in a poster format at the 2008 Experimental Biology/American Association for Nurition Meetings in San Diego, CA.
PARTICIPANTS: The individuals who participated in the project were Kristen Scheckel, Undergraduate student, who received an Undergraduate Research Training Award as part of the Undergraduate Research Program; Stephanie Degner, who received her Ph.D. in Nutritional Sciences; Corrina Fuentes, undergraduate student and lab assistant; Donato F Romagnolo, Associate Professor and Principal Investigator.
TARGET AUDIENCES: Target audiences for this work include medical and nutrition professionals, graduate students and medical practitioners, and the general public. Specific targets include individuals who may have increased susceptibility to colon and breast cancer, and are interested in learning more about how dietary agents can help in preventing inflammatory processes that increase the risk of colon and breast cancer.
PROJECT MODIFICATIONS: No project modifications are necessary
Impacts
Mechanisms of prevention of COX-2 activation by the dietary agent rosmarinic acid. One mechanism through which bioactive food components may exert anti-cancer effects is by reducing the expression of the proinflammatory gene cyclooxygenase-2 (COX-2), which has been regarded as a risk factor in tumor development. Rosmarinic acid (RA) is a phenolic derivative of caffeic acid present in rosemary (Rosmarinus officinalis). Previous research documented that RA may exert anti-inflammatory effects. However, the mechanisms of action of RA on COX-2 expression have not been investigated. We report that in colon cancer HT-29 cells, RA reduced the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity and protein levels. In HT-29 and breast cancer MCF-7 cells, the cotreatment with RA reduced TPA-induced transcription from a control AP-1 promoter-luciferase construct and binding of the AP-1 factors c-Jun and c-Fos to COX-2 promoter oligonucleotides harboring a
cAMP-response element (CRE). The anti-AP1 effects of RA were also examined in a non-malignant breast epithelial cell line (MCF10A) in which RA antagonized the stimulatory effects of TPA on COX-2 protein expression, the recruitment of c-Jun and c-Fos to the COX-2/CRE oligonucleotides, and activation of the extracellular signal-regulated protein kinase-1/2 (ERK1/2), a member of the mitogen-activated protein kinase (MAPK) pathway. Thus, we propose that RA may be an effective preventative agent against COX-2 activation by AP-1 inducing agents in both cancer and non-malignant cells.
Publications
- Scheckel KA, Degner SC, Romagnolo DF. 2008. Inhibition of Cyclooxygenase- 2 Expression by Rosmarinic Acid Through Repression of AP-1 in Colon and Breast Cell Lines (Journal of Nutrition, Submitted)
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Progress 01/01/06 to 12/31/06
Outputs
Environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to act as endocrine disruptors and tumor promoters. In previous studies, we documented that estrogen (E2)-induced BRCA-1 transcription required the recruitment of an AP-1/ER complex to the proximal BRCA-1 promoter. Here, we report that activation of BRCA-1 transcription by E2 requires occupancy of the BRCA-1 promoter by the unliganded aromatic hydrocarbon receptor (AhR). The stimulatory effects of E2 on BRCA-1 transcription are counteracted by 1) cotreatment with the AhR antagonist 3'methoxy-4'-nitroflavone (3M4NF); 2) transient expression in ER-negative Hela cells of ER lacking the protein-binding domain for the AhR; and 3) mutation of two consensus xenobiotic responsive elements (XRE=5'-GCGTG-3') located upstream of the ER binding region. These
results suggest that the physical interaction between the unliganded AhR and the liganded ER plays a positive role in E2-dependent activation of BRCA-1 transcription. Conversely, we show that the AhR ligands B[a]P and TCDD aborgate E2-induced BRCA-1 promoter activity. The repressive effects of TCDD are paralleled by increased recruitment of the liganded AhR and HDAC1, and reduced occupancy by p300 and SCR-1 at the BRCA-1 promoter region flanking the XREs. We propose that the ligand status of the AhR modulates transcriptional activation of the BRCA-1 promoter by estrogen. In a separate study, we investigated the effects of CLA on COX-2 transcription in breast cancer cells. Results of transient transfection studies revealed that treatment of breast cancer MCF-7 cells with a CLA mix or selected isomers (c9, t11-CLA; t10, c12-CLA) at concentrations ranging from 20 to 80 mol/L, attenuated in a dose-dependent fashion COX-2 transcription induced by the proinflammatory agent
12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, the CLA mix inhibited TPA-induced activity of the human collagenase-1 promoter. Because TPA activates the activator protein-1 (AP-1) transcription factor, we investigated the effects of CLA on recruitment of nuclear proteins to a cAMP response element (CRE) (-59/-53) in the COX-2 promoter and a consensus TPA-responsive element (TRE) in the human collagenase-1 promoter, which are binding sites for AP-1 members. Using electrophoretic mobility shift assays, we found that the CLA mix reduced TPA-induced recruitment of nuclear proteins to the COX-2 CRE and human collagenase-1 TRE. Binding studies revealed that the t10, c12-CLA isomer was more effective than the CLA mix or c9, t11-CLA in reducing binding of cJun to either the COX-2 CRE or human collagenase-1 TRE, whereas LA increased binding to both elements. Overexpression of the AP-1 member, c-Jun, reversed the inhibitory effects of the CLA mix on COX-2 transcription, and restored
binding of nuclear proteins to the CRE and TRE. We conclude that the ability of CLA to inhibit AP-1 may contribute to its effectiveness as an anti-carcinogenic and anti-inflammatory agent.
Impacts
This work will impact our understanding of the dietary risk factor in the etiology of sporadic breast cancer and inflammation and the potential role of preventative dietary agents
Publications
- Kemp MQ, Thorne PA, Liu W, Kane MD, Selmin O, and Romagnolo DF. 2006. Expression Profiles Induced by Polycyclic Aromatic Hydrocarbons: The Transferrin Receptor as an Aromatic Hydrocarbon Receptor-inducible Biomarker of Exposure. Environmental Molecular Mutagenesis 47:518-526.
- Hockings J.K., Thorne P.A., Kemp M.Q., Morgan S.S., Selmin O., Romagnolo D.F. 2006. The Ligand Status of the Aromatic Hydrocarbon Receptor Modulates Transcriptional Activation of BRCA-1 Promoter by Estrogen. Cancer Research, 66:1-9.
- Degner S.C., M.Q. Kemp, Bowden GT, D.F. Romagnolo. 2006 Conjugated Linoleic Acid Attenuates Cyclooxygenase-2 Transcriptional Activity via an Anti-AP-1 Mechanism in MCF-7 Breast Cancer Cells. J. Nutr.136:421-427.
- Romagnolo D.F., S.C. Degner, M.Q. Kemp, J.K. Hockings, and O. Selmin. 2006. Role of dietary xenobiotics-gene interactions in carcinogenesis: protective effects of nutritional factors. Current Nutrition Review&Food Science. 2:205-214.
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Progress 01/01/05 to 12/31/05
Outputs
Diet is a vehicle of genotoxic agents, which have been shown to be carcinogenic in experimental models. Our efforst have focused on tumorigenicity induced by xenobiotics such as PAHs that may interfere with expression of tumor suppressor genes such BRCA-1 and p53. Under normal conditions, these tumor suppressors play a key role in control of cell cycle progression and DNA repair. Dysregulation of these genes may increase genomic instability predisposing to tumor growth. We also examined the overlapping effects of PAHs and dioxins on expression of P450s and COX-2 and the role of dietary fatty acids as risk modifiers by influencing the production of proinflammatory and mutagenic endproducts. Finally, we investigated the potential benefits of polyphenolic and synthetic compounds as antagonists of the activated aromatic hydrocarbon receptor (AhR). Whereas the exposure to dietary xenobiotics may be practically unavoidable, future investigations should examine the effects
of synergistic interactions between nutrients and dietary xenobiotics on expression and function of tumor susceptibility genes and the physiological role of protective nutritional factors. These efforts may lead to the development of combinatorial dietary interventions to retard and possibly prevent cancer progression.
Impacts
Interactions between dietary xenobiotics and nutrients influence cancer risk. Epidemiological studies in humans suggested that diet is an important vehicle of exposure to various xenobiotics, including polycyclic aromatic hydrocarbons (PAHs), dioxins, and chlorinated hydrocarbons (CHs). The activation of the aromatic hydrocarbon receptor (AhR) pathway by PAHs and dioxins stimulates the expression of several genes including cytochrome P450s, which metabolize PAHs to highly mutagenic compounds that cause fixation of mutations in the p53 gene and repress the expression of the tumor suppressor gene, BRCA-1. Conversely, PAHs and dioxins have been shown to activate the expression of cyclooxygenase-2 (COX-2), whose protein product participates in the production of reactive PAH-metabolites and synthesis of proinflammatory prostaglandins (PGs). The carcinogenicity of CHs has been attributed to their ability to activate the expression of oncogenes including c-myc, and fatty
acid activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma). Dietary components that may protect against the activation of the AhR pathway include flavonoids, which comprise a large family of dietary phenolic phytochemicals found in fruits and vegetables. Research indings produced in our laboratory document the protective effects of natural (genestein) and synthetic (alpha-naphthoflavone) flavonoids against PAH-induced changes in gene expression.
Publications
- Romagnolo D.F., S.C. Degner, M.Q. Kemp, J.K. Hockings, and O. Selmin. 2006. Role of dietary xenobiotics-gene interactions in carcinogenesis: protective effects of nutritional factors. Current Nutrition Review&Food Science. In Press.
- Hockings J.K., Thorne P.A., Kemp M.Q., Morgan S.S., Selmin O., Romagnolo D.F. 2006. The Ligand Status of the Aromatic Hydrocarbon Receptor Modulates Transcriptional Activation of BRCA-1 Promoter by Estrogen. Cancer Research, 66:1-9.
- Degner S.C., M.Q. Kemp, and D.F. Romagnolo. 2006 Conjugated Linoleic Acid Attenuates Cyclooxygenase-2 Transcriptional Activityvia an Anti-AP-1 Mechanism in MCF-7 Breast Cancer Cells. J. Nutr. 136:421-427.
- Jeffy B.D., Hockings J.K., Kemp M.Q., Morgan S.S., Hager J.A., Beliakoff J., Whitesell L.J., Bowden G.T., Romagnolo D.F. 2005. An Estrogen Receptor-alpha/p300 Complex Activates the BRCA-1 Promoter at an AP-1 Site That Binds Jun/Fos Transcription Factors: Repressive Effects of p53 on BRCA-1 Transcription. Neoplasia. Sep;7(9):873-82.
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Progress 01/01/04 to 12/31/04
Outputs
The environmental carcinogen benzo[a]pyrene (B[a]P) is a prototype polycyclic aromatic hydrocarbon (PAH) found in foods and tobacco smoke, and ligand of the aromatic hydrocarbon receptor (AhR). Conversely, flavonoids compete with PAHs for binding to the AhR. In this study, we wished to characterized in breast epithelial cells the toxicogenomic responses induced by B[a]P and the protective effects of the synthetic flavonoid and AhR-ligand alpha-naphthoflavone (ANF). Acute exposure of breast cancer MCF-7 cells to B[a]P induced a number of changes in physiological state including S-phase arrest, segregation of nucleolar material, and disruption of ribosomal transcription. These changes were antagonized by cotreatment with ANF. Using a cDNA microarray approach, we examined the effects of B[a]P and ANF on the mRNA profile of selected categories of genes encoding for members of the P450 family, DNA damage and repair, and cell cycle regulation. Transcripts that were
upregulated by B[a]P included members of the cytochrome P450 family (CYP1A1, CYP1B1, CYP2J2), and cell cycle regulators (cdc28 protein kinase-2). In contrast, B[a]P downregulated transcript levels of glutathione G-transferases (GSTT1, GSTA2, GSTM2), cyclins (A2, D1, and E1), cdc25C, and DNA repair genes including PARP and RCC5. Treatment with ANF alone increased GSTM2 mRNA whereas ANF plus B[a]P tended to restore normal expression profiles. The S-phase arrest induced by B[a]P was accompanied by accumulation of p53 and p21 protein. Conversely, the co-treatment with ANF restored normal cell cycle distribution and prevented the accumulation of p53 and p21. These results highlight important cellular and molecular effects of B[a]P which may be useful biomarkers of exposure to PAHs and the potential usefulness of flavonoids as antagonists of dietary and environmental AhR ligands.
Impacts
Identify environmental and dietary risk factors in the onset on carcinogenesi of the breast and colon
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Role of Bile Acids and PAHs in Carcinogenesis of the Colon. This project deals with the role of bile acids in the onset of colon carcinogenesis, interactions between bile acids and dietary carcinogens and molecular changes that may predispose to the onset of colon cancer. Deoxycholate, a bile salt present at high levels in the colonic lumen of individuals on a high fat diet, is a promoter of colon cancer. Deoxycholate also causes DNA damage. BRCA1 functions in repair of DNA and in induction of apoptosis. We show that when cultured cells of colonic origin are exposed to deoxycholate at different concentrations, BRCA1 expression is induced at a low non-cytotoxic concentration (10 uM), but is strongly inhibited at higher cytotoxic concentrations (100 uM). Indication of phosphorylation of BRCA1 by DOC (100 uM) at a lower dose was seen by Western Blot analysis, while at a higher dose, DOC (200 and 300 uM) caused a complete loss of BRCA1 expression. We show that BRCA1 is
substantially lower in colon adenocarcinomas from five patients compared to associated non-neoplastic colon tissue from the same patients, suggesting that the loss of BRCA1 expression contributes to the malignant phenotype. In the non-neoplastic colon tissue, BRCA1 was localized to the non-goblet cells. Transcriptional and Post-transcriptional Changes Induced by the Carcinogen Benzo[a]pyrene: Effects of the Synthetic Flavonoid alpha-naphthoflavone. The objective of this study was to characterize in breast epithelial cells the toxicogenomic responses induced by benzo[a]pyrene, a prototype polycyclic aromatic hydrocarbon (PAH) and ligand of the aromatic hydrocarbon receptor (AhR), and the B[a]P-metabolite 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). Acute exposure of breast cancer MCF-7 cells to B[a]P induced S-phase arrest, loss of membrane integrity, and ultrastructural changes in cellular architecture including segregation of nucleolar material and
disruption of nucleolar fibrillar centers. As a means to assess global changes in gene expression induced by PAHs, we analyzed by cDNA microarray the temporal effects of B[a]P, BPDE, and the AhR agonist/antagonist alpha-naphthoflavone (ANF) on the mRNA levels of 1134 genes (GeneMAPTM CancerArray) subdivided in 16-functionally diverse categories. Transcripts that were upregulated by B[a]P included members of the cytochrome P450 family (CYP1A1, CYP1B1, CYP2J2), cell cycle regulators (cdc28 protein kinase-2), histone 3A (H3F3A), chromatin structure (SWI/SNF), peroxisome proliferator activated receptor-gamma (PPARG)and retinoic acid receptor-alpha (RARA). In contrast, B[a]P downregulated transcript levels for various cyclins (A2, D1, and E1), IGFBP-3, Bcl-2, AP-1 (JunD and Fos), fibroblast growth factor receptor1 (FGFR1), and the estrogen receptor-alpha (ESR1). The treatment with BPDE triggered genotoxic responses similar to those elicited by B[a]P, but at lower doses and earlier time
points, whereas the co-treatment with ANF tended to counteract the effects of B[a]P on cell cycle arrest, cellular ultrastructure, and mRNA profiles.
Impacts
Identify environmental and dietary risk factors in the onset on carcinogenesi of the breast and colon
Publications
- Michael Kemp, Ryan B. Chirnomas, Brandon D. Jeffy Ramya Ranganathan, Nisha Sahay, Michael D. Kane, and Donato F. Romagnolo. Toxicogenomics of exposure to polycyclic aromatic hydrocarbons: protective effects of the synthetic alpha-naphthoflavone. In submission, Journal of Nutrition, also presented at the 95th Annual Meeting of the American Asociation for Cancer Research, 2004.
- Romagnolo DF, Chirnomas RB, Ku J, Jeffy BD, Payne CM, Holubec H, Ramsey L, Bernstein H, Bernstein C, Kunke K, Bhattacharyya A, Warneke J, Garewal H. Deoxycholate, and endogenous tumor promoter and DNA damaging agent, modulates BRCA-1 expression in apoptosis-sensitive epithelial cells: loss of BRCA-1 expression in colonic adenocarcinomas. Nutrition and Cancer 46:82-92, 2003
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Progress 01/01/02 to 12/31/02
Outputs
The objective of this study was to characterize in breast epithelial cells the toxicogenomic responses induced by benzo[a]pyrene, a prototype polycyclic aromatic hydrocarbon (PAH) and ligand of the aromatic hydrocarbon receptor (AhR), and the B[a]P-metabolite 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). Acute exposure of breast cancer MCF-7 cells to B[a]P induced S-phase arrest, loss of membrane integrity, and ultrastructural changes in cellular architecture including segregation of nucleolar material and disruption of nucleolar fibrillar centers. As a means to assess global changes in gene expression induced by PAHs, we analyzed by cDNA microarray the temporal effects of B[a]P, BPDE, and the AhR agonist/antagonist alpha-naphthoflavone (ANF) on the mRNA levels of 1134 genes (GeneMAPTM CancerArray) subdivided in 16-functionally diverse categories. Transcripts that were upregulated by B[a]P included members of the cytochrome P450 family
(CYP1A1, CYP1B1, CYP2J2), cell cycle regulators (cdc28 protein kinase-2), histone 3A (H3F3A), chromatin structure (SWI/SNF), peroxisome proliferator activated receptor-gamma and retinoic acid receptor-alpha. In contrast, B[a]P downregulated transcript levels for various cyclins (A2, D1, and E1), IGFBP-3, Bcl-2, AP-1 (JunD and Fos), fibroblast growth factor receptor-1 (FGFR1), and the estrogen receptor-alpha. The treatment with BPDE triggered genotoxic responses similar to those elicited by B[a]P, but at lower doses and earlier time points, whereas the co-treatment with ANF tended to counteract the effects of B[a]P on cell cycle arrest, cellular ultrastructure, and mRNA profiles. Changes in the cDNA microarray profiles of a subset of genes following exposure to B[a]P and BPDE were validated by RT-PCR analysis.
Impacts
Overall, these cumulative data were consistent with previous reports documenting the antiproliferative and endocrine-disrupting effects of AhR-ligands. The current studies suggest that acute exposure to PAHs may induce specific adaptive responses in cellular architecture and expression profile, which may be useful in developing predictive biological models of exposure to PAHs.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The decontamination of aflatoxin B1 (AFB1)-contaminated corn using an integrated ammoniation fermentation process was studied. Different concentrations [0.25, 0.50, 1.00, 1.5, and 2.0% (w/w)] of ammonium persulfate were tested in the detoxification of AFB1 contaminated corn during fermentation. In order to increase the ethanol production during fermentation and decontamination of corn, 0.5 and 1.0 % (w/w) of azodicarbonamide, benzoyl peroxide, or hydrogen peroxide were tested. Peroxides were added at three different stages of the fermentation process: liquefaction, saccharification, and fermentation. Levels of AFB1 and ethanol were determined after every fermentation process. Treated corn was tested for mutagenic potential using the Salmonella/microsomal mutagenicity assay with TA 100 tester strain and pure AFB1 as positive control. The greater level of decontamination was obtained after the addition of 2.0% (w/w) ammonium persulfate without any effect on the ethanol
production. The addition of peroxides did not significantly increase the ethanol production or significantly improve the decontamination process. The best process for decontamination of corn included the addition of 2.0% (w/w) ammonium persulfate and 1.0% (w/w) benzoyl peroxide. The best process for ethanol production was the addition of 2.0% (w/w) ammonium persulfate and 0.5% (w/w) benzoyl peroxide. No treated corn samples showed mutagenic potential.
Impacts
This process could be used for the production of fuel ethanol from aflatoxin containing corn and rendering the by-product, distillers dried grains and solubles, free from aflatoxin.
Publications
- DECONTAMINATION OF AFB1-CONTAINING CORN BY AMMONIUM PERSULFATE DURING FERMENTATION.Armando Burgos-Hernandez, Ralph L. Price, Karen Jorgensen-Kornman, Rebeca Lopez-Garcia, Henry Njapau, and Douglas L. Park. 2002 Journal of the Science of Food and Agriculture. Accepted. In press
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Progress 01/01/00 to 12/31/00
Outputs
Treatment of whole and ground corn naturally contaminated with 200 ppb total aflatoxin with stabilized granulated ammonium bicarbonate at 25 or 37 degrees, at 10, 15, or 17.5% moisture levels for 0 to 20 days with samples taken at 5 day intervals was unsuccessful in lowering levels of the mycotoxin under any of the conditions described. We are continuing the study using regular powdered ammonium bicarbonate and subjecting the samples to the same testing parameters. We expect that release of ammonia will be more rapid and destruction of the aflatoxin will be similar to our previous extrusion experiments.
Impacts
Continued investigation into methods of elimination of aflatoxin and fumonisin from already-contaminated commodities may provide an economical means to avoid the destruction of that product and at the same time assure safety to those consuming it.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
We are following the production of fumonisin in culture to see if it is first produced and then destroyed by substances within the culture itself. If so, we want to isolate the substances responsible. Four flasks containing sterile nutrient broth were inoculated with a suspension of a fumonisin- producing strain of Fusarium moniliforme. All were held at 25 degrees C, two flasks with agitation and two without. Samples taken from the flasks for each of 27 days were analyzed for fumonisin. Fumonisin was detectable in the agitated flasks after 3 days with maximum levels at 7 days. Then fumonisin was gradually reduced until day 14 after which a drastic reduction took place. This would indicate that formed fumonisin was being destroyed by some factor in the media, perhaps by the aged mold culture. Cultures in the non-agitated flask and in flasks with moistened, inoculated corn meal and whole corn did not produce fumonisin under our experimental conditions. Whole and ground
corn were treated with granulated ammonium bicarbonate at 25 or 37 degrees, at 10, 15, or 17.5% moisture levels for 0 to 20 days with samples taken at 5 day intervals. This study was to find a simple, inexpensive way to treat high moisture corn on the farm while awaiting the drying process. Samples were extracted with either water/methanol (20/80)or 0.1 N HCl/methanol (20/80) to verify the complete destruction of the aflatoxin. Treatment of the whole grain were largely unsuccessful in reducing aflatoxin. We are analyzing the ground corn. It is possible that our use of a stabilized ammonium bicarbonate, normally used for soil treatment, did not allow the development of sufficient ammonia to penetrate the kernels.
Impacts
Continued investigation into methods of elimination of aflatoxin and fumonisin from already-contaminated commodities may provide an economical means to avoid the destruction of that product and at the same time assure safety to those consuming it.
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
We are attempting to monitor the production of fumonisin in synthetic culture and in corn to see if it is first produced and then destroyed by substances within the culture itself as is aflatoxin. If this is so, we want to isolate the substances responsible. Four flasks containing sterile nutrient broth were inoculated with a suspension of a fumonisin-producing strain of Fusarium moniliforme. All were held at 25 degrees C, two flasks with agitation and two without. Samples taken from the flasks for each of 27 days were analyzed for fumonisin. Fumonisin was detectable in samples taken from the agitated flasks after 3 days with maximum levels at 7 days. After that levels were gradually reduced until day 14 after which a drastic reduction took place. Fumonisin levels were close to zero for the rest of the experiment. This would indicate that formed fumonisin was being destroyed in the latter part of the experiment by some factor in the media, perhaps by the aged mold
culture. Cultures in the non-agitated flask and in flasks with moistened, inoculated corn meal and whole corn did not produce fumonisin under our experimental conditions
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/97 to 12/31/97
Outputs
The project under this objective is in its beginning stages. We are attempting to monitor the production of fumonisin in synthetic culture to see if it is first produced and then destroyed by substances within the culture itself as is aflatoxin. If this is so, we want to isolate the substances responisible. We have been collecting preliminary information, learning HPLC techniques for fumonisin analysis and trying to obtain a fumonisin producing strain of Fusarium. A survey of commercially produced corn-based snack products has shown no fumonisin present.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/96 to 12/30/96
Outputs
Fumonisin (FB1)-contaminated corn during nixtamalization w/ Ca(OH)2 treated w/ NaHCO3 + H2O2, w/ NaHCO3 or w/ H2O2 alone, gave 100% reduction of FB1. Brine shrimp mortality w/ 0.25 g FB1 /reaction products dropped from 40% (untreated corn extract) to 17% (chemically-treated corn). FB1 concentrations of 0.0025-100 g/plate showed a small pos. mutagenicity. No newly formed mutagens were detected in treated samples. Untreated corn extract exhibited positive mutagenicity; subsequent analysis showed 65 ppb of aflatoxin B1 which would account for the above mutagenicity. Aflatoxin-contaminated corn containing 140 g/kg was fermented with 2% ammonium persulfate and H2O2, azocarbonamide, or benzoyl peroxide added at 2 concentrations (0.5 or 1%)at different stages (liquefaction, saccharification & fermentation) of the process.The most significant aflatoxin decrease was in samples treated with 2% ammonium persulfate and 0.5% benzoyl peroxide (97%). The highest ethanol production
was found in samples treated with 2% ammonium persulfate with 0.5% benzoyl peroxide added during saccharification just prior to fermentation inoculation.The same treatment with 1% benzoyl peroxide inhibited ethanol production.All samples tested negative using the Salmonella/mammalian microsomal mutagenicity assay with TA100. Samples of the original corn combined with pure AFB1 standard showed a decrease in mutagenic activity. This suggests the presence of compound(s) in corn which interfere with the mutagenic activity of aflatoxin.
Impacts
(N/A)
Publications
- PARK, D,L., LOPEZ-GARCIA, R., TRUJILLO-PRECIADO, S., and PRICE, R.L. Reduction of Risks Associated with Fumonisin Contamination in Corn. In: Fumonisins in Food, Ch. 29, Jackson, L.
- S., DeVries, J. W. and Lloyd, B. Bullerman (Ed.) PlenumPres.
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Progress 01/01/95 to 12/30/95
Outputs
Nixtamalization of fumonisin B1 (FB1 )-contaminated corn using Ca(OH)2 and NaHCO3 + H2O2 or H2O2 alone gave 100% reduction of FB1. Brine shrimp mortality with FB1/reaction products dropped from 40% to 17%. No newly formed mutagens were detected in the treated samples. Fumonisin B1 in acetic acid/sodium acetate buffer was destroyed by NaOH, NH4OH, and (NH4)2S2O8 and decreased by hydrogen (96%) and horseradish peroxide (70%). Toxicity of samples to brine shrimp was equal to or greater than that of the fumonisin control. Selected Arizona cotton fields were monitored for A. flavus contamination at different heights of the cotton plant and for meteorological factors using the AZMET program. Aflatoxin was determined in immature cotton bolls and cottonseed prior to harvest and in modules. There were higher levels of A. flavus on lower portions of the plant. Aflatoxin contamination was 5 times higher on insect damaged cotton bolls than on normal bolls. High relative humidity
and rainfall favored A. flavus contamination. Aflatoxin-contaminated roasted peanuts cooked in water with hydrogen peroxide in combination with sodium or ammonium bicarbonate, sodium hydroxide, and ammonium persulfate had aflatoxin reduced by half by cooking alone. None of the chemicals alone or in combination with hydrogen peroxide were successful in eliminating aflatoxin from the peanut sauce.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
Glutathione (GSH) added to the Salmonella mutagenicity assay decreased the mutagenic potential of aflatoxin B1 (AFB1) AFB2 and AFB1:AFB2 mixtures to TA100. S9 mix converted about 5% of AFB2 to AFB1. Aflatoxins were broken down by lactoperoxidase (LP) and horseradish peroxidase (HRP)with oxygen present, with low H202 concentrations (0.1-1%), but not in the absence of oxygen or H202. In vitro fumonisin B1 was reduced 70-85% by treatment with 0.01M Ca(OH)2, 3% H202 , and 2% Na2CO3 alone or in combinations for 0.5 to 12 hours at 23C. Optimum breakdown was with a combination of all reagents, but including peroxide in the reaction mixture had little addtional effect. Brine shrimp and microbiological assays confirmed the reduction of fumonisin. Extracts from aflatoxin- free corn and aflatoxin- contaminated corn with and without ammonia treatment, tested for mutagenic potential using the Ames test (TA 100) gave evidence of an "anti aflatoxin mutagenic factor" in corn that may
block the mutagenic activity of aflatoxin in the Ames test.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Naturally-contaminated, roasted peanuts containing with 2600 ppb total aflatoxin(AF) were ground and cooked with water, H(subscript 2)O(subscript 2), NaHCO(subscript 3), NH(subscript 4)HCO(subscript 3), or ammonium persulfate. Cooking alone reduced AF by half. Although cooking with food additives further reduced AF, no chemical alone or in combination with H(subscript 2)O(subscript 2) were successful in eliminating AF. Naturally-contaminated corn containing 500 ppb total AF was fermented using commercial procedures to determine fate of aflatoxin and alcohol production. H(subscript 2)O(subscript 2), NaOH, NH(subscript 4)OH, NH(subscript 4)Cl, ammonium persulfate, catalase, and horseradish peroxidase alone or in combinations were added during fermentation. Alcohol production was lower with NH(subscript 4)Cl but higher when catalase and horseradish peroxidase were used together. High levels of H(subscript 2)O(subscript 2) produced slightly more alcohol but slowed or
inhibited fermentation. Aflatoxin was reduced in DDGS of samples treated with either ammonium or sodium hydroxide. H(subscript 2)O(subscript 2), catalase, and horseradish peroxidase alone caused a reduction in AF; greatest reductions occurred with the combination of H(subscript 2)O(subscript 2), catalase, and peroxidase. Ammonium persulfate destroyed nearly all aflatoxin, but completely inhibited production of alcohol.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Naturally-contaminated corn with 500 ppb and in-shell peanuts with 280 ppb totalaflatoxin (AF) with 10, 15, 17.5 or 20% moisture were stored in plastic bags at 21(degree), 32(degree), or 43(degree) C for 5, 10, 20, or 40 days. There was no increase in AF in corn with moisture of 10, 15 or 17.5%. There was an increase in toxin after 10, 20 or 40 days at 20% moisture with levels dependent upon temperature. AF in corn stored at higher temperatures decreased after 20 days. AF in peanuts stored in the shell increased under all storage conditions. Analysis of tortillas made from the above corn with 0.75% Ca(OH)(subscript 2) and 0, 0.1, 0.5, or 1% ammonium bicarbonate during cooking indicated that those made using ammonium bicarbonate had less AF than those which did not. No process decreased the AF to below the 20 ppb level except for the 1% ammonium bicarbonate sample (13 ppb). The above corn was fermented using NaOH or NH(subscript 4)OH to increase the pH to 9.6 during
liquefaction. H(subscript 2)O(subscript 2), lactoperoxidase and/or horseradish peroxidase were added during the process. Fermentation with NH(subscript 4)OH showed significant decreases in AF which were even greater with the peroxide and peroxidases. Fermentation with NaOH was unsuccessful in reducing AF to below 20 ppb.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
All experiments used field corn, naturally-contaminated with 400 ppb aflatoxin (AF). Corn meal was extruded from an X-5 Wenger extruder at temperatures above 100(degree)C with 16% added moisture and combinations of H(subscript 2)O(subscript 2) and NH(subscript 4)OH or NH(subscript 4)HCO(subscript 3). Extrusion alone permanently destroyed about 50% of the AF. Only NH(subscript 4)OH or NH(subscript 4)HCO(subscript 3) reduced AF below 20 ppb. The odor of ammonia could not be sensed on the product afterwards. Neither the original or treated samples produced the expected mutagenicity with the Ames test. No destruction of amino acids by extrusion under alkaline conditions was evident. Corn meal was treated with 1 or 2% H(subscript 2)O(subscript 2) and 1% NaOH with and without preheating (75(degree)C) for 30 min. All treatments were equally successful in permanently lowering the AF by 90%. In no case was AF decreased to below 20 ppb. Tortillas were made from whole corn was
cooked for 1.25 h in 0.75% Ca (OH)(subscript 2) (w/w) and allowed to soak 15 hr. Also 0.1, 0.5 or 1% ammonium bicarbonate was added. Using increasing ammonium bicarbonate resulted in lowered AF. No process decreased the AF to below 20 ppb. Whole corn in plastic bags in 500 g lots with 10, 15, 17.5, and 20% total moisture was stored at 70, 90, and 110(degree)C for 5, 10, 20, and 40 days. AF increased drastically only in samples stored at 20% moisture; after 20 days it decreased to below original levels.
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Progress 01/01/90 to 12/30/90
Outputs
Milk obtained from cows fed rations containing aflatoxin-contaminated cottonseed, ammonia-treated aflaxtoxin-contaminated cottonseed, and uncontaminated cottonseed were tested for mutagenic potential using the Ames Salmonella assay. Samples included whole milk, nonfat dry milk powder, cream, and reconstituted whole milk. High mutagenic activity was seen in samples from contaminated cottonseed but not in samples from cows fed the ammonia treated, contaminated cottonseed. Field corn, naturally-contaminated with 400 ppm aflatoxin was extruded above 100C with 16% added moisture and combinations of calcium hydroxide, hydrogen peroxide, sodium bicarbonate, ammonium hydroxide, sodium bisulfite, ammonium bicarbonate, azodicarbonamide, and benzoyl peroxide. Extrusion alone destroyed approximately 50% of the aflatoxin. Ammonium hydroxide and ammonium bicarbonate reduced aflatoxin below 20ppb. Changes in aflatoxin B(1) and B(2) mutagenic potentials were determined using the
Salmonella assay with and without S9 liver homogenate and glutathione (GSH). Addition of GSH to the assay showed a decrease in the mutagenic potential for AFB(1), AFB(2) and mixtures. GSH without S9 eliminated the mutagenic potentials of AFB(1) and AFB(2). The AFB(1):AFB(2) mixture of 1:0.3 most closely resembled activity of AFB(1) alone.
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Progress 01/01/89 to 12/30/89
Outputs
Aflatoxin contaminated whole cottonseed was treated with ammonia using two commercial methods, a high pressure, high temperature process (14% moisture, 4% ammonia gas, 100C, 40 lbs/in, 30 min, 1200 mu g/Kg) and an atmospheric pressure, ambient temperature process (1 1/2% anhydrous ammonia, 15% water, ambient temperatures, 42 days, 5200 mu g/Kg). Rations containing the treated seed were fed to dairy cows (15 and 25% of the ration, respectively), and milks obtained were dried and incorporated into trout (Salmo gairdneri) diets (25%). In the pressure method, aflatoxin M(1) in the untreated seed/milk trout diets was 8 mu g/Kg. No tumors nor neoplastic lesions were observed in livers of trout fed milk from either the ammonia-treated or untreated source. In the ambient method, 18% of the trout consuming milk from cows with non-ammoniated cottonseed in the ration had liver tumors; those consuming milk from cows with ammonia-treated aflatoxin-contaminated cottonseed in their
rations had no tumors nor neoplastic lesions. These data demonstrate that the ammonia process reduces risks posed by aflatoxin contamination.
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Progress 01/01/88 to 12/30/88
Outputs
Mutagenicity of terminal aflatoxin B(subscript 1) and B(subscript 2) precursors were determined using Ames test (TA 98) in rat liver homogenate. O-Methylsterigmatocystin (OMST) and AFB, exhibited some cytotoxicity to the tester strain. Sterigmatocystin and OMST exhibited positive responses. 1 ug OMST is required to show same response exhibited by 0.02 u2 AFB(subscript 1). AFB(subscript 2) and precursor did not show activity. Maximum levels tested were 45 ug/plate for dehydro-OMST, and 100 ug/plate for hydroxy-OMST and AFB(subscript 2). Effects of glutathione with and without AFB(subscript 2) on AFB(subscript 1) mutagenic potential in the Ames test and TA 100 was determined with and with S9. There was a decrease in potential of both AFB(subscript 2) and AFB(subscript 1), when glutathione was added to S9. AFB(subscript 2) had no effect on potential of AFB(subscript 1) in concentration of 0.3:1. With the 1:1 effect as additive. For plates which contained neither
glutathione nor S9, there was a decrease in potential and cytotoxicity. AFB(subscript 1) - contaminated cottonseed (1500 ppb) was ammoniated and fed to cows as 25% of ration. Milk (3 ppb aflatoxin M(subscript 1)) was spray-dried for trout studies. Milks were tested for potential using Salmonella/ microsome mutagenicity strains TA 98 and TA 100. Samples from cows exposed to aflatoxin cottonseed and the spiked milks showed high levels of activity.
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Progress 01/01/87 to 12/30/87
Outputs
Jojoba albumins and globulins were treated with jojoba tannins to study the protein-tannin complex formation. Trypsin and chymotrypsin were also treated with jojoba tannins to determine the change of enzymatic activity. The treated albumins and globulins were fractionated by gel filtration before and after treatment. Two albumin fractions did not appear after treatment but a new fraction appeared with a shorter elution time. A precipitate was formed by addition of jojoba tannins to the jojoba globulins and two globulins fractions did not appear after treatment. Trypsin was completely inhibited by 5.80 mu g of tannin/ml. Chymotrypsin was completely inhibited by 36.44 mu g of tannin/ml. Whole cottonseed, naturally-contaminated with AFB(1) was ammoniated commercially and fed as 25% of a dairy ration to 3 cows. The contaminated whole seed was likewise fed. The cream was removed from the milk of these cows, the skim milk was spray-dried and the powder from both
treatments was sent to Oregon State for evaluation of carcinogenicity with rainbow trout.
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Progress 01/01/86 to 12/30/86
Outputs
Naturally-contaminated yellow dent corn containing 1,500 mu g total aflatoxin per kg was treated with 2.5 or 3% NaOH at 100C for 2 or 5 min respectively. After washing, the moisture was adjusted by soaking or autoclaving and the product was deep fried for 10 or 15 min to produce a corn snack. There was greater than 99% reduction of the aflatoxin in the final product. Acidifying products prior to analysis caused reformation of as much as 18% of the original aflatoxin; the acidified final product showed no reformation. Mutagenesis detected by Salmonella typhimurium (TA 98 and TA 100) decreased as aflatoxin levels decreased. Mutagenicity of the final product was equal to that of the negative controls. Naturally-contaminated spray dried milk was added to raw or pasteurized whole milk to a final concentration of 1.1 mu g AFM(1)/1. Formalin (37% w/w) was added to these milk solutions to give concentrations of zero, .025, .05 and 0.1% formaldehyde, and samples were
stored in the dark at 21C in either plastic or glass containers. Samples were analyzed for AFM(1) at 0, 1, 2, 3 and 4 weeks. A second trial used only raw milk and glass containers with AFM(1) analyses at 0, 1, and 2 weeks. Twenty-five percent less AFM(1) was detected in milk stored in plastic than in glass containers, but whether the milk was raw or pasteurized had no effect. AFM(1) losses increased during storage and with increasing formaldehyde concentration. AFM(1) in milk preserved with 0.
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Progress 01/01/85 to 12/30/85
Outputs
The effects of glutathione (GSH) and the combination of GSH and glutathione-S-transferase (G-S-T) on aflatoxin B(1) (AFB(1)) mutagenesis using Salmonella typhimurium strains TA 98 and TA 100 were tested. Ten concentrations of AFB(1) (0 to 1.0 ug/plate) were added to a liver microsomal homogenate (S9 mix) and incubated for 0 and 30 minutes at 37 C prior to plating. One third of the samples contained GSH and one third contained G-S-T. Of the samples incubated prior to plating one half contained bacteria. Addition of GSH and GSH+G-S-T decreased both mutagenic activity and lethality of AFB(1) to the bacteria. Formation of the AFB(1)-GSH conjugate was verified using reverse phase HPLC. A simplified procedure for treating AFB(1) contaminated, whole cottonseed in the seed handling system at the gin was tested. Water and anhydrous ammonia were metered into the pneumatic seed handling systems at 10 and 1.5% respectively. Samples taken from the gin stream every 10 minutes
for 7 hr each day for 3 days and from each pile after the entire run and at 24, 48, 72, and 96 hr intervals indicated no reduction of AFB(1). Yellow corn which was naturally-contaminated with 1570 ppb of total aflatoxin was treated with 2.5 or 3% NaOH for 2 or 5 min. After washing and moisture adjustment the product was deep fried for 10 or 15 min. Both manufacturing processes were effective in reducing aflatoxin in the finished product to nearly non-detectable levels and mutagenic activity to background.
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Progress 01/01/84 to 12/30/84
Outputs
The effectiveness of cottonseed ammoniation for removal of carcinogenic substances from milk from cows consuming aflotoxin-contaminated whole cottonseed as a part of their diet was determined. Milk was obtained from cows which had consumed aflatoxin-contaminated whole cottonseed (5000 mu g/kg) or the same cottonseed which had been treated with 11/2% anhydrous ammonia and 10% water at ambient temperatures and pressure. These two samples along with appropriate controls were included as 20% of the diet of Rainbow trout for 9-12 months. No hepatomas were found in 2 groups of 100 trout consuming regular trout rations, a control diet containing 20% normal non-fat dried milk, or a diet containing 20% milk from cows consuming ammoniated cottonseed. Hepatomas (26%) were found in trout consuming a control diet with 4 mu g/kg aflatoxin B(1), and in trout (18%) consuming a diet containing 20% milk from cows consuming non-treated, contaminated cottonseed. Aflatoxin M(1) has
about 1/3 the carcinogenicity of AFB(1) to trout: ammoniation of the cottonseed reduces carcinogneicity of the trout diets to that of the control. Ninety high producing dairy cows were fed aflatoxin-contaminated whole cottonseed as 15% of their diet for 70 days. Aflatoxin levels in their milk indicated that approximately 1.6% of the dietary aflatoxin is excreted in the milk. Six dairy cows were fed aflatoxin, ammoniated aflatoxin or ammoniated aflatoxin which had been acidified.
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Progress 01/01/83 to 12/30/83
Outputs
Five processes for tortilla manufacture all decreased aflatoxin (AF) from naturally contaminated corn up to 88%; however, acidifying all products prior to analysis caused reformation of up to 44% of the original aflatoxin. Mutagenicity of the acetone extracts was also decreased by tortilla manufacturing processes. Treatments of aflatoxin-contaminated peanut meal with NaHSO(3), H(2)O(2), and NaOC1 with or without 1% Ca(OH)(2) were all effective in reducing aflatoxin levels at 0.5 and 0.2% concentration. Alkaline media greatly reinforced the effectiveness of the oxidizing agents. All treatments were effective in decreasing mutagenic response of S. typhimurium TA 98 and TA 100 strains. Complete elimination of AFB(1) or mutagenicity was not obtained under these treatment conditions. Comparison of results obtained from chemical analyses with those obtained with the Ames test gave a correlation coefficient of 0.9. Direct HPLC injection of the ammonia-AFB(1) reaction
media indicated the rapid formation of two very polar by-products. These compounds were stable at pH 12 but reverted quickly to AFB(1) during mass spectral analysis.
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Progress 01/01/82 to 12/30/82
Outputs
Naturally contaminated peanut meal was treated with various chemicals at different concentrations (0, 0.5, and 2.0%) and different temperatures (22 and 62 degrees C), and aflatoxin degradation was measured. All treatments, NaHSO(3), H(2)O(2) plus 1% Ca (OH)(2), NaOCl, and NaOCl plus l% Ca (OH)(2), were effective in decreasing the amount of aflatoxin and the mutagenic response of S. typhimurium TA98 and TA100 strains. Adding 1%Ca(OH)(2) to NaCCl decreased mutagenic activity significantly; whereas adding it to H(2)O(2) did not. Of all treatments, NaOCl plus 1%Ca(OH)(2) was the most effective; nevertheless complete elimination of aflatoxin and of mutagenicity of extracts was not achieved. The higher temperature caused a significant decrease of both aflatoxin and mutagenicity. Comparison of analytical results with results of the Ames test gave a correlation coefficient of 0.9. Crystalline aflatoxin B(1) was treated with concentrated aqueous ammonia at 25 degrees C
under aerobic and anaerobic conditions. Samples were removed periodically and tested for residual aflatoxin and reaction products. At least two reaction products with a greater polarity than aflatoxin B(1) were formed. During identification attempts using mass spectrometry, these compounds reverted to aflatoxin B(1). Temperatures higher than 60 degrees C during ammoniation were necessary to cause changes in aflatoxin B(1) which were not reversible under acidic conditions.
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Progress 01/01/81 to 12/30/81
Outputs
Experimental shrimp, Penaeus stylirostrus and P. vannamei, were exposed to 2.0 to 160mg aflatoxin B(1) per kg body weight by intramuscular injection or by multiple per os dosing with feed (53 to 300 mg aflatoxin B(1)/kg feed) for up to 24 days. Histopathogenesis of aflatoxicosis in the exposed animals was time and dose-dependent in the hepatopancreas, mandibular organ, and in the hematopoietic organs. Inconsistant lesions were observed in other organs and tissues. Aflatoxin B(1) was added in various concentrations to skim milk inoculated with three strains of Lactobacillus and three strains of Streptococcus in pure and mixed cultures. Skim milk contaminated naturally with 0.6 Mug aflatoxin M(1)/1 was inoculated with the same lactic acid bacteria. Aflatoxin B(1) caused an increase in the chain length of Streptococcus bacteria but caused no significant changes in morphology nor in coagulation time of the milk. Aflatoxin M(1) delayed coagulation of skim milk by over
33 hours.
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Progress 01/01/80 to 12/30/80
Outputs
Aflatoxin-contaminated cottonseed was treated with 1-1/2% ammonia and 10% water and packed into 10 foot diameter by 100 foot long polyethene bags and held for 21 days. The ammoniated cottonseed was fed to lactating dairy cattle in two different trials. Analysis of milk for aflatoxin M(1) showed a maximum level of 1.8 ppb from the group receiving the untreated cottonseed and a maximum of 0.8 from the group receiving the ammoniated meal. Both groups returned to negative 6 days after cottonseed feeding ceased. Dried citrus waste was fed to dairy cows and their milk was analyzed according to the standard methods for AFM(1). Results indicate that a compound from the citrus waste may interfere with the analysis of aflatoxin M(1) in milk by HPLC. This interference can be overcome by several methods. A short-term toxicity study of aflatoxin B(1) on Benaeid shrimp involving three major routes of contact: 1) intramuscular injection, b) ingestion, and c) absorption through
the gills indicated that 0.600 mg aflatoxin B(1) per kg feed did not significantly increase the mortality rate even after three weeks. In addition, little cellular damage was observed. Injection of 51 mg aflatoxin B(1) per kg body weight caused severe damage and mortality after 5 days. No damage was observed in the hepatopancreas from doses less than 2 mg aflatoxin B(1) per kg body weight. There was little lethality due to absorption of aflatoxin B(1) at 10 mg/L seawater.
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Progress 01/01/79 to 12/30/79
Outputs
1. Reduction of aflatoxin M(1) (90%) in milk from cows fed whole cottonseed contiminated with 680 ppb aflatoxin B(1) (AFB(1)) was achieved by treatment of the seed with 1 l/2% aqueous ammonia and 10% added H(2)O. Ammoniation was carriet out in a 10X8X100 ft. long polyethylene bag using an "Ag bagger". 2. Distruction of AFB(1) in whole cottonseed atmospheric pressure was determined at 0, 1, 1.5 and 2% aqueous ammonia, 7.5, 15, 17.5 and 20% moisture, at 70, 90 and 110 degrees F, for 5, 10, and 15 days. The most rigorous treatment reduced AFB(1) from 800 ppb to below 10 ppb. 3. Acute and subacute toxicity effects to AFB(1) upon Peneaid shrimp showed an approximate LD(50) of 30-40 Mug/g in 24 hours and 15-20 Mug/g in 96 hours. Histological effects were observed in the feeding (subacute) study. 4. During separation of AFM(1) naturally contaminated milk into fractions, more than 50% remainder in the whey. Upon ultrafiltration of the whey, 75% of the AFM(1) remained in
the permeate. Upon separation of the proteins, 86% of the AFM(1) was lost, 14% remained with the globulin fraction and none followed the albumin fraction.
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Progress 01/01/78 to 12/30/78
Outputs
1) Samples of Arizona commercial milk were shown to have levels of aflatoxin M(1) above 1(ppb) during July and August. Cause was determined to be a large lot of extremely contaminated cottonseed which had entered the dairy feed chain. Analysis of milk produced previous and subsequent to these months had insignificant amounts of aflatoxin (less than 15% of dairies with less than or equal to 0.1 ppb). 2) During ultrfilitration of cheese whey, aflatoxin M(1) from naturally contaminated milk was concentrated in the whey protein fraction. An unknown aflatoxin metabolite which interferes with Af M(1) analysis in both TLC and HPLC has been isolated and determined not be Af M(1), M(2), nor B(2a). R(f) values also eliminate other known Af metabolites. Attempts at identification are continuing. 4) Examination of peanuts, peanut products, and milk from commercial sources near Guaymas, Sonora, Mexico showed all peanut candy and 80% of the peanut butter to exceed 20 ppb
aflatoxin B(1). Whole and canned peanuts had much lower levels, and no aflatoxin M(1) was found in milk samples.
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Progress 01/01/77 to 12/30/77
Outputs
Thin layer chromatography was used to separate resorcinol compounds, and Rf values were determined for several wheat and triticale grain samples. Different Rf values were found for wheat and triticale. Jojoba meal was fed to weanling mice at two levels for either 4 or 6 weeks. The mice were removed from the feed, raised to sexual maturity, and mated. Mice fed jojoba meal for 6 weeks at both dietary levels had reduced numbers of pups born and weaned. Paired mice fed at a rate of equal body weight had no impairment of reproduction. Aflatoxin M(1) was found in detectable levels (=0.1 ppb) in 10-15% of bulk tank milk samples collected on the bimonthly basis from 154 different dairies affitrated with United Dairymen of Arizona (UDA). Testing of a large number of dairy feed ingredients in Arizona has implicated cottonseed and cottonseed products as the sources of aflatoxin for contamination of milk. Dairy cows from the University of Arizona herd were fed varying levels of
aflatoxim and milk was weighed and tested for aflatoxin. No loss in milk production was observed at the levels fed. Tests of 100 cows from each of five Arizona dairies suggest that cows within a herd being fed the same contaminated mix do not secrete aflatoxin in the milk at the same levels. Evidence points to feed selection and not to differences in metabolism of the aflatoxin by the individual cow. Attempts to produce antibodies to patulin by injecting rabbits with patulin conjugated to ficoll or to epoxysepharose were unsuccessful.
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Progress 01/01/76 to 12/30/76
Outputs
Weanling mice were fed four diets for a period of three weeks and then placed onmouse chow. The dietary treatments included controls, 5% jojoba meal, 10% jojoba meal and purified simmondsin being added at a level equal to the 5% jojoba meal. At maturity, the mice were mated and reproduction records kept. The incorporation of either jojoba meal or purified simmondsin in the diet of weanling mice for a period of three weeks caused reduction in number of pups born and weaned. The isolated simmondsin had results similar to the 5% jojoba meal treatment. The simmondsin of jojoba meal affects the reproduction in mice. The naturally occurring toxin, resorcinol, of grain is being tested chemically for structure. The resorcinol has been demonstrated to lower ME value of grain samples. Grain samples with approximately equal resorcinol concentrations do not have similar ME values. These differences may be do to different resorcinol compounds.
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Progress 01/01/75 to 12/30/75
Outputs
The simmonds in toxin of the jojoba bean was isolated and purified. The purified crystals of simmondsin are to be added to whole egg control diets and fed to weanling mice for a period of three weeks. The mice are to be placed on laboratory chow and mated for a reproduction study. Various grain samples have been tested for resorcinol concentrations. In addition, the standard curve usedin the resorcinol method is being investigated. Future plans call for the use of thin-layer chromatography to identify the various types of resorcinol compounds present.
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Progress 01/01/74 to 12/30/74
Outputs
Jojoba meal was fed at levels of 0, 5 and 10% of the diet by replacing equal amounts of soybean meal and resulted in reduced growth in weanling mice at the 5and 10% level. After the initial four weeks the mice were placed on laboratory chow and paired for breeding purposes. The initial mating of mice resulted in 9, 5 and 2 pups for the average number born to the 0, 5 and 10% treated mice. The additional crossing of the different treated males and females resulted in reduced litter size by the 5 and 10% treated female mice. Resorcinol values were determined for 23 varieties of wheat, 5 varieties of triticale, 6 varietiesof sorghum and 5 varieties of barley grains. Correlation studies are under way to determine the relationship between resorcinol levels of the grain and their protein quality plus metabolizable energy values.
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Progress 01/01/73 to 12/30/73
Outputs
A series of three feeding experiments were run with jojoba meal in both weanlingand young adult mice. The jojoba meal, when fed as the sole source of protein in the diet, resulted in 100% mortality after a two week period. The jojoba meal was autoclaved, and/or suppelmented in various combination, to correct the deficient amino acids (methioine, lysine and isoleucine). Jojoba meal fed to young adult mice caused a reduction in feed intake by about 60% and resulted in a weight loss of 35%. The isolated wax from the jojoba bean was fed to both weanlings and young adult mice and resulted in a 50% reduction in growth plus a 30% mortality in weanling mice. Adult mice fed a jojoba diet for a one week period had a 20% reduction in weight plus 40% reduction in feed intake, but no mortality observed.
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