Progress 10/01/14 to 09/30/19
Outputs
Target Audience:The primary target audience is the scientific community, in the academic, government and private sectors. This includes students striving for college/university degrees in the agricultural and life sciences at the undergraduate and graduate levels. All these individuals are served by the contribution of this project to the knowledge base in the area of health-promoting dietary agents delivered by foods. A secondary target audience is the general public who may be counseled on dietary choices by nutrition educators. We plan to disseminate knowledge to the targeted audiences through presentations at professional and technical meetings, campus meetings and seminars, classroom instruction, and ultimately publications in appropriate peer-reviewed journals. The secondary audience will most likely be reached through interviews or other exchanges with project principles with writers representing magazines as well as other popular print and digital media. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A post-doctoral associate working on the project was responsible for one-on-one mentoring and training of a Ph.D. and two undergraduate students assisting on this project. Through individual study and partnering with collaborating labs, further skill development for the PhD student was realized in the areas of molecular biology and the conduct of in vivo animal trials. How have the results been disseminated to communities of interest?Inter-group presentations were arranged among three collaborating lab groups in Food Science. The post-doc reported locally and at scientific meetings on research progress. Audience members included graduate students, academic staff and faculty in the participating labs, all of which have active research programs focused on food, diet and health. Four scientific journal accounts of our results were published from this Grant, and another is anticipated after the conduct of an animal trial (which will occur after this grant expires). Also, an M.S. thesis was produced and deposited in the UW-Madison library. The intended audience for all of these activities is principally scientists and researchers at all levels of professional development who seek new knowledge in areas related to the scope of the project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The anticipated impact of this project is to provide scientific validation of how specific types of sulfur compounds in Allium vegetables (e.g. onion, garlic), when included in the diet, are effective at mitigating disease-linked inflammatory processes, using a high-fat diet to induce obesity in mice. During this reporting period we have elucidated details by which Allium sulfur compounds attenuate inflammatory signaling in macrophage cells, subject to inflammatory signaling pathway by lipopolysaccharide (LPS) activation of the nuclear factor-kappa B (NF-κB) signaling pathway, a fairly universal model of inflammation. A second potentially beneficial effect evaluated was to determine the ability of these sulfur compounds to elevate cellular antioxidant defenses to protect against oxidative stress and determine the mechanism behind this action. The dietary trial was initiated at the end of the final project year, but the outcome will be determined by mid-2020. Allium vegetable-derived metabolites, S-alk(en)ylmercaptocysteine (CySSR: R = allyl, "A" from garlic; or 1-propenyl, "Pe" from onion), were prepared from tissues blends and purified on a Reveleris Prep purification system using a Discovery C18 column. Pre-treatments with CySSR conferred concentration-dependent reductions in cytokines (TNF-α, IL-1β and IL-6), NO production and iNOS (inducible nitric synthase) overexpression, and attenuated oxidant production in LPS-stimulated macrophage cells (viability remained> 90%). These protective effects were manifested through inhibited activation of the NF-κB signaling pathway via suppression of the IκB kinase (IKK) phosphorylation. The attenuation of LPS-induced inflammation by CySSRs was associated with enhanced levels of cellular cysteine (CySH) and glutathione (GSH) mediated by cellular import/reduction of CySSR and the induction of glutamate cysteine ligase (GCL), one of >200 nuclear factor erythroid 2-related factor 2 (Nrf2) regulated proteins. The reduction of anti-inflammatory effect by CySSR was implicated to involve enhanced levels of GSH, likely in the context of Nrf2-regulated enzymes that scavenge H2O2 and peroxides using GSH as co-substrate. The anti-inflammatory effect of CySSPe was significantly greater than CySSA for almost all indicators measured, and cell metabolites of CySSRs may have a role in attenuating NF-κB signaling. These results will assist in identifying suitable molecular targets to assess for in vivo efficacy of anti-inflammatory effects of CySSR. The onion-derived metabolite, S-1-propenylmercaptocysteine (CySSPe), protects against oxidative stress and exhibits anti-inflammatory effects by modulating cellular redox homeostasis. We sought to establish whether CySSPe activates nuclear factor erythroid 2-related factor 2 (Nrf2) and whether activation of Nrf2 by CySSPe involves modification of the Kelch-like ECH-associated protein-1 (Keap1) to manifest these effects. We found that CySSPe stabilized Nrf2 protein and facilitated nuclear translocation to induce expression of antioxidant enzymes, including NQO1, HO-1, and GCL. Moreover, CySSPe attenuated tert-butyl hydroperoxide-induced cytotoxicity and dose-dependently inhibited reactive oxygen species production. Silencing experiments using Nrf2-siRNA confirmed that CySSPe conferred protection against oxidative stress by activating Nrf2. CySSPe enhanced cellular pool of reduced glutathione (GSH) and improved GSH:GSSG ratio. Pretreatment of cells with L-buthionine- S,R-sulfoximine (BSO) confirmed that CySSPe increases de novo synthesis of GSH by upregulating expression of the GSH-synthesizing enzyme GCL. Treatment of cells with CySSPe elevated hydrogen sulfide (H2S) production. Inhibition of H2S-synthesizing enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), by pretreating cells with propargylglycine (PAG) and oxyaminoacetic acid (AOAA) revealed that H2S production was partially dependent on a CSE/CBS-catalyzed β-elimination reaction with CySSPe that likely produced 1-propenyl persulfide (RSSH). Depleting cells of their GSH pool by exposure to BSO and diethylmaleate attenuated H2S production, suggesting a GSH-dependent formation of H2S, likely via the reduction of RSSH by GSH. Finally, treatment of cells with CySSPe persulfidated Keap1, which may be the mechanism involved for the stabilization of Nrf2 by CySSPe. Taken together, our results showed that attenuation of oxidative stress by CySSPe is associated with its ability to produce H2S or RSSH, which persulfidates Keap1 and activates Nrf2 signaling. This study provides insights on the potential of CySSPe as an onion-derived dietary agent that modulates redox homeostasis and combats oxidative stress. An animal trial (mice) was initiated the month after this project expired, where the onion-derived metabolite, S-1-propenylmercaptocysteine (CySSPe) was administered in high-fat diets to model development of obesity. The feeding period ended 31 December 2019 and analyses will be conducted over the next several months to document the effects of CySSPe in mice relative to subjects w/o CySSPe supplementation.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Jiang, H. Preparation and solubility of mixed-disulfide conjugates of protein and Allium thiosulfinates, M.S. Thesis, University of Wisconsin-Madison
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Tocmo, R. and K.L. Parkin (2018). S-Alk(en)ylmercaptocysteine suppresses LPS-induced pro-inflammatory responses in murine macrophages through inhibition of NF-kB pathway and modulation of thiol redox status. Free Radic. Biol. Med. 129:548-558.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Tocmo, R. and K.L. Parkin (2018) Data on chromatographic isolation of cysteine mixed-disulfide conjugates of Allium thiosulfinates and their role in cellular thiol redox modulation, Data in Brief, 21:1445-1450.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Qiu-R-Y. Wang, J-R. and K.L. Parkin (2018). Activity-guided isolation of phase II enzyme inducers from buckwheat flour methanolic extracts. J. Sci Food Agric. 98:4911-4918.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Tocmo, R. and K.L. Parkin (2019). S-1-propenylmercaptocysteine protects murine hepatocytes against oxidative stress via persulfidation of Keap1 and activation of Nrf2. Free Radic. Biol. Med. 143:164-175.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The primary target audience is the scientific community, in the academic, government and private sectors. This includes students striving for college/university degrees in the agricultural and life sciences at the undergraduate and graduate levels. All these individuals are served by the contribution of this project to the knowledge base in the area of health-promoting dietary agents delivered by foods. A secondary target audience is the general public who may be counseled on dietary choices by nutrition educators. We plan to disseminate knowledge to the targeted audiences through presentations at professional and technical meetings, campus meetings and seminars, classroom instruction, and ultimately publications in appropriate peer-reviewed journals. The secondary audience will most likely be reached through interviews or other exchanges with project principles with writers representing magazines as well as other popular print and digital media. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A post-doctoral associate working on the project was responsible for one-on-one mentoring and training of a Ph.D. and an undergraduate student assisting on this project. The sophomore undergraduate student will continue to assist on this project this next year. Through individual study and collaboration with collaborating labs, further skill development for project personnel was realized in the areas of molecular biology and the conduct of in vivo animal trials. How have the results been disseminated to communities of interest?Inter-group presentations were arranged among three collaborating lab groups in Food Science. The post-doc reported on research progress. Audience members included graduate students, academic staff and faculty in the participating labs, all of which have active research programs focused on food, diet and health. Two journal accounts of our results were published in 2018, plus a third contribution primarily supported by a visiting doctoral student with home institutional funding. The intended audience for all of these activities is principally scientists and researchers at all levels of professional development who seek new knowledge in areas related to the scope of the project. What do you plan to do during the next reporting period to accomplish the goals?Going forward, based on a pilot animal trial this past, we will modify the animal model for IDB that reduces variability among subjects in each experimental group. Animal studies will make use of CySSR (instead of protein-conjugates) because we can quantitatively control for the composition/proportion of R groups as 1-propenyl or allyl residues. None of these adjustments are outside the scope of the original proposal/project.
Impacts
What was accomplished under these goals?
The anticipated impact of this project is to provide scientific validation of how specific types of sulfur compounds in Allium vegetables (e.g. onion, garlic), when included in the diet, are effective at mitigating disease-linked inflammatory processes. Two such inflammation-mediated diseases are colitis and obesity. During this reporting period, we have elucidated details by which Allium sulfur compounds attenuate inflammatory signaling in macrophage cells, subject to inflammatory signaling pathway by lipopolysaccharide (LPS) activation of the nuclear factor-kappa B (NF-κB) signaling pathway, a fairly universal model of inflammation. Allium vegetable-derived metabolites, S-alk(en)ylmercaptocysteine (CySSR: R = allyl, "A" from garlic; or 1-propenyl, "Pe" from onion), were prepared from tissues blends and purified on a Reveleris Prep purification system using a Discovery C18 column. Pre-treatments with CySSR conferred concentration-dependent reductions in cytokines (TNF-α, IL-1β and IL-6), NO production and iNOS (inducible nitric synthase) overexpression, and attenuated oxidant production in LPS-stimulated macrophage cells (viability remained> 90%). These protective effects were manifested through inhibited activation of the NF-κB signaling pathway via suppression of the IκB kinase (IKK) phosphorylation. The attenuation of LPS-induced inflammation by CySSRs was associated with enhanced levels of cellular cysteine (CySH) and glutathione (GSH) mediated by cellular import/reduction of CySSR and the induction of glutamate cysteine ligase (GCL), one of >200 nuclear factor erythroid 2-related factor 2 (Nrf2) regulated proteins. The reduction of anti-inflammatory effect by CySSR was implicated to involve enhanced levels of GSH, likely in the context of Nrf2-regulated enzymes that scavenge H2O2 and peroxides using GSH as co-substrate. The anti-inflammatory effect of CySSPe was significantly greater than CySSA for almost all indicators measured, and cell metabolites of CySSRs may have a role in attenuating NF-κB signaling. These results will assist in identifying suitable molecular targets to assess for in vivo efficacy of anti-inflammatory effects of CySSR.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Tocmo, R. and K.L. Parkin (2018). S-Alk(en)ylmercaptocysteine suppresses LPS-induced pro-inflammatory responses in murine macrophages through inhibition of NF-kB pathway and modulation of thiol redox status. Free Radic. Biol. Med. 129:548-558.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Tocmo, R. and K.L. Parkin (2018) Data on chromatographic isolation of cysteine mixed-disulfide conjugates of Allium thiosulfinates and their role in cellular thiol redox modulation, Data in Brief, 21:1445-1450.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Qiu-R-Y. Wang, J-R. and K.L. Parkin (2018). Activity-guided isolation of phase II enzyme inducers from buckwheat flour methanolic extracts. J. Sci Food Agric. 98:4911-4918.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The primary target audience is the scientific community, in the academic, government and private sectors. This includes students striving for college/university degrees in the agricultural and life sciences at the undergraduate and graduate levels. All these individuals are served by the contribution of this project to the knowledge base in the area of health-promoting dietary agents delivered by foods. A secondary target audience is the general public who may be counseled on dietary choices by nutrition educators. We plan to disseminate knowledge to the targeted audiences through presentations at professional and technical meetings, campus meetings and seminars, classroom instruction, and ultimately publications in appropriate peer-reviewed journals. The secondary audience will most likely be reached through interviews or other exchanges with project principles with writers representing magazines as well as other popular print and digital media. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A post-doctoral associate working on the project was responsible for one-on-one mentoring and training of a Ph.D. and an undergraduate student assisting on this project. The undergraduate student in currently in graduate school working in a similar area as this project (bioactive food agents). Through individual study and collaboration, skill development for project personnel was realized in the areas of molecular biology and the conduct of in vivo animal trials. How have the results been disseminated to communities of interest?A graduate seminar presentation was given by a Ph.D. student on this project to report on research progress toward the first major goal to date. Audience members included: graduate students, academic staff and faculty in Food Science as well as individuals from allied departments showing an interest in this work. Another similar presentation was given by a post-doctoral associate to a group meeting (undergraduate & graduate students, post-docs and faculty) involving two laboratories on the background leading to addressing the second major goal of this project. What do you plan to do during the next reporting period to accomplish the goals?We will continue the work on both objectives.
Impacts
What was accomplished under these goals?
The anticipated impact of this project is to provide scientific validation of how specific types of sulfur compounds in Allium vegetables (e.g. onion, garlic), when included in the diet, are effective at mitigating disease-linked inflammatory processes. Two such inflammation-mediated diseases are colitis and obesity. We have developed the technology to attach Allium sulfur compounds to common food proteins in a manner that retains anti-inflammatory effect. Using proteins as vehicles to deliver these active agents through common food formulations or as dietary supplements provides the incentive and means for individuals to potentially reduce risk of inflammatory diseases with only modest adjustment to dietary habits. We have established the mechanism by which Allium sulfur compounds attenuate inflammatory signaling in cells. A major and universal inflammatory pathway is suppressed and the levels of specific pro-inflammatory chemicals are reduced. The significance of these observations is that it provides indicators or "marker chemicals" that can be tested to show whether the Allium sulfur compounds are effective at reducing inflammation when included in the diet of mice subjected to inflammatory stress. If these compounds are effective in mice, there is optimism that they will also be effective in humans. Thus, this year, the key accomplishment was the generation of knowledge in how Allium sulfur compounds inhibit inflammation in cells, providing a suitable foundation for designing feeding trials to assess how efficacious these compounds are in ameliorating inflammatory in living organisms.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Food Science professionals and graduate students, as well as, other technical personnel on UW-Madison campus who attended a graduate seminar reporting progress on this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training and professional development has been afforded to the graduate student in the field of food protein chemistry and bioactive food components using cultured cells as a model. Other facets of student training include making progress toward meeting requirements of the graduate degree programs at UW-Madison, including coursework, research mentoring and counsel provided by meetings with the students' Graduate Program Advisory Committee. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Scale up preparation of Prot-SSR to support animal trials. Identify responses of cultured cells to CySSR and cell thiols (CySH and GSH) in terms of H2S generation and anti-inflammatory responses. Start conducting animal feeding trials to determine efficacy of protein conjugates in reducing risk of disease.
Impacts
What was accomplished under these goals?
We have completed our target goal (#1) of developing an understanding of how to pretreat food proteins to attain predictable degrees of SH group exposure, focusing on WPI and egg white protein (EWP). The next step is to conjugate pretreated proteins with Allium thiosulfinates. The resulting Protein-SSR conjugates will be produced on a scale sufficient to conduct animal trials with fixed doses of the presumptively active group (CySSR). The purpose is to determine physiological responses to Protein-SSR in the context of providing any in vivo benefit in models of chronic inflammation. We have also identified a potential mediator of biological responses to CySSR (besides CySSR itself) - that being H2S. Appropriate cellular responses to CySSR and H2S will be incorporated into the in vivo trials after further cellular studies are completed.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Food Science professionals and graduate students, as well as, other technical personnel on UW-Madison campus who attended a graduate seminar reporting progress on this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training and professional development has been afforded to the graduate students participating in this project. The general area of training is in the field of food protein chemistry. More specific focus of training involves protein structure, and how perturbing structure can facilitate exposure and enhanced reactivity of amino acid side chains. The students were also involved with the pilot scale extraction of juice from Allium tissue using the same type of screw press that is used by onion processing plants where commercial waste is used to generate energy. The modification of protein for delivering health benefits of Allium tissue would make use of such a unit operation in a viable commercial process if the outcomes of this project are adapted by the commercial sector. Other facets of student training includes making progress toward meeting requirements of the graduate degree programs at UW-Madison, including coursework, research mentoring and counsel provided by meetings with the students' Graduate Program Advisory Committee. How have the results been disseminated to communities of interest?As part of a student's graduate program requirements, a formal seminar was given on campus at UW-Madison Food Science Department that disseminated research results that were generated during the reporting year of the project. What do you plan to do during the next reporting period to accomplish the goals?1) Scale up preparation of protein mixed disulfide conjugates to support in vivo trials. 2) Start to conduct animal feeding trials to determine efficacy of protein conjugates in reducing risk of disease.
Impacts
What was accomplished under these goals?
We have successfully addressed our target goal (#1) of SH group exposure in WPI to advance to the next step of conjugation modified WPI with Allium thiosulfinates. Presuming a reasonable yield of 90% conjugation, this would yield sufficient levels of mixed disulfide conjugates to furnish the dosing needs for the subsequent in vivo trials, pending scale up of the protein conjugate preparations.The next immediate step is to scale-up preparation of WPI-conjugates to conduct animal feeding trials. This is a prerequisite to designing and executing the feeding trial experiments.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2015
Citation:
Preparation and solubility of mixed-disulfide conjugates of protein and Allium thiosulfinates
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