Are NonVitamin A Active Carotenoid Cleavage Products Metabolically
Are Non-Vitamin A Active Carotenoid Cleavage Products Metabolically Active? John W. Erdman Jr. , Ph. D Department of Food Science and Human Nutrition University of Illinois at Urbana Champaign
Outline • Norman Krinsky remembered • Metabolism of vitamin A and non-vitamin A active carotenoids • Lycopene metabolism in mice lacking carotenoid cleavage enzymes • Impact of lycopene/tomato powder on androgen metabolism in mice lacking CMO-I or CMO-II • Conclusions
Goals of presentation What I hope to do • Introduce a broad audience to mammalian metabolism of carotenoids • Provide foundation for several other talks • Make a case for biological activity of metabolites of lycopene What I will not do • Review the extensive work of others • Review tomato/lycopene and prostate cancer
Norman Krinsky (1928 -2008)
Norman Krinsky (1928 -2008) • Gentleman scientist • Loved people and travel • Preached the Scientific Method without preaching • Mentor to hundreds of scientists • Wonderful way of asking questions • Warm heart and rich mind
Norman Krinsky (1928 -2008) • Initiated 1 st Gordon Conference on Carotenoids in 1992 • 56 years in carotenoid research • Postdoctoral Fellow of George Wald • Worked on over 30 different carotenoids • Chaired the DRI panel on Antioxidants • Received over 30 professional honors
Thanks Norm
Chemical Structures of Major Carotenoids in Human Blood (Lobo et al, 2011)
Metabolism of pro-vitamin A carotenoids 1. β-carotene (and α-carotene, β-cryptoxanthin, etc. ) is the source of retinal and retinol. 2. CMO-I is a cytosolic enzyme whose best known function is to convert carotenoid precursors into vitamin A 3. Further oxidation of retinol/retinal can yield retinoic acid, ligands for RAR and RXR 4. CMO-I also is a critical molecular player for the regulation of PPARγ activity in adipocytes (Amergual, et al, Plo. S One, 2011) and other perhaps tissues
β-Carotene Metabolism in cells expressing both BCMO 1 (CMO-I) and BCDO 2 (CMO-II) (Lobo et al, 2011)
Classic Retinoic Acid Function Retinoic Acid: Regulates numerous transcription factors
Metabolism of carotenoids 1. While physiological levels of β-carotene are cleaved by CMO-I, lycopene, for example, is primarily cleaved by CMO-II, not by CMO-I 2. CMO-II is a mitochondrial enzyme that apparently is responsible for degrading excess carotenoids and reduce potential toxicity of these molecules (von Lintig talk on Weds) 3. Products of CMO-I and II are further metabolized /degraded by a variety of enzymes
Underlining Hypotheses Hypothesis - Some non-provitamin A carotenoid metabolites are bioactive The focus of the presentation will mostly be on lycopene and other tomato carotenoids
Biogenesis of Lycopene
Lycopene metabolites LYCOPENOIDS are defined as metabolites of lycopene that may have biological effects in a manner similar to RETINOIDS Lindshield et al, (2007) Arch Biochemistry & Biophysics
Cleavage of tomato carotenoids ? CMO-I: Carotenoid monooxygenase I, carotene-15, 15’-monooxygenase, BCMO 1… CMO-II: Carotenoid monooxygenase II, carotene-9’ 10’-monooxygenase, BCDO 2…
In vitro studies Cell culture studies have provided clues to specificity of CMO-I and II for carotenoids.
e. coli model β-carotene Accumulating + CMO-II β-carotene Accumulating + CMO-I lycopene Accumulating + CMO-II lycopene accumulating β-carotene accumulating Kiefer, et al. 2001
Studies with knock-out mice Creation of mice lacking either CMO-I or CMO-II have proved extremely valuable in discerning the importance of these enzymes in carotenoid and lipid metabolism These mice can help answer the question of whether lycopene is metabolized by CMO-II in vivo
CMO I KO Mice Wild-type CMO I KO 20
Journal of Nutrition 138: 2367 -71(2008) Loss of Carotene-9’, 10’-monooxygenase expression increases serum and tissue lycopene concentrations in lycopene-fed mice Nikki Ford, Steven Clinton, Johannes von Lintig, Adrian Wyss & John Erdman Journal of Nutrition 140: 2134 -2138 (2010) 21
Hepatic lycopene • CMO-I KO • CMO-II KO • Wild-type Ford, et al. , J Nutr (2010)
Testes lycopene • CMO-I KO • CMO-II KO • w. WTw. Tldtype Ford, et al. , J. Nutr. (2010)
What is the source of lycopenoids in tissues? • CMO-II produces lycopenoids in a variety of mammalian tissues • Kopec et al (J. Ag. Food Chem. 2010) identified and quantified apo-lycopenals in fruits, vegetables and in human plasma (Schwartz Tuesday) • Evidence suggests that the source of lycopenoids in the body are from foods and from metabolism
Are lycopenoids bioactive? • Sharoni, Levy, and colleagues have provided early in vivo evidence for bioactivity (see lectures by both on Tuesday) • X-D Wang, et al, have suggested that apolycopenoic acid protects against cancer (see lecture on Tuesday) • The Erdman lab has focused on lycopenoid effects on androgen status and PPARγ
Bioactivity of Lycopene Metabolites An ethanolic extract of stored lycopene containing lycopene metabolites and oxidation products was as effective as lycopene itself in activated the antioxidant response element in cancer cells. Ben-Dor et al, (2005) Mol. Cancer Therapeutics
Effect of ethanolic extract of lycopene or lycopene on ARE transactivation Ben-Dor, A. et al. Mol Cancer Ther 2005; 4: 177 -186
Bioactivity of Lycopene Metabolites Lycopene was added to media for a 72 hour treatment period or added with new media every 24 hours for 72 hours and proliferation of DU 145 human prostate cells was monitored. (Ford et al, Nutrition and Cancer (2011)
Interrelationship between lycopene androgens Does reduction in androgen status impact tissue accumulation of lycopene? Does increasing dietary feeding of tomato powder or lycopene have an impact on androgen status?
What is the impact of androgen status upon lycopene metabolism? The influence of castration, with or without testosterone implants, or food restriction upon tissue levels of lycopene was tested in F 344 rats. Boileau et al, (2000) J. Nutr. 130: 1613 -8. Boileau et al, (2001) J. Nutr. 131: 1746 -52.
Outline
What is the impact of androgen status upon lycopene metabolism? - Castrated rats consume 80% of the food of rats with free access to food - Castrated rats accumulate twice the hepatic lycopene as rats with free access to food
What is the impact of androgen status upon lycopene metabolism? The results showed that androgen depletion or 20% food restriction increased hepatic lycopene accumulation. Similar findings have been reported for vitamin E (Feingold et al BBA 1176: 192 -6; 1993). Higher androgen status and greater consumption of energy may stimulate lycopene metabolism and degradation.
Inverse question • We have shown that lower androgen status result in higher tissue lycopene concentrations • What about the reverse question – the impact of more lycopene on serum and tissue androgens? Results in rats Results in KO mice
Carotenoids androgen status Oral dose (0. 7 mg/day) Surgery 57 d 59 d 62 d Sacrifice 63 d Control n=8 Sham Phytofluene n=8 Lycopene F 344 Rats (N=80) n=8 fed Control Powder Diet AIN-93 G 10% Tomato Powder n=8 35 d age Control n=12 Campbell et al J Nutr. 2006 Castrated Phytofluene n=12 Lycopene n=12 10% Tomato Powder n=12
Serum Testosterone Concentrations following 4 Day Carotenoid Feeding or Castration a b b b, c c c p < 0. 05
Serum DHT Concentrations following 4 Day Carotenoid Feeding or Castration a a b b p < 0. 05
Unpublished Study GOAL = Determine whether lycopene metabolism affects sex steroid status in wild type or mice lacking one of the two carotenoid cleavage enzymes. (Ford et al, in submission )
Study Design Wild Type CMO-II KO CMO-I KO 1 week washout AIN-93 G 4 Day Feeding Tomato Powder AIN-93 G Lycopene Beadlet Placebo Beadlet • Wild-Type (C 57 BL/6 Jx 129 x 1/Sv. J) 29 -32 weeks of age • Tomato Powder diet had 204 nmol/g diet and Lycopene beadlet diet had 248 nmol/g diet
Lycopenoid concentrations and half-life • Plasma levels of lycopenals = nmol/L • Tissue levels of lycopenals = pmol/g • These are in line with plasma and tissue levels of retinoids like retinoic acid • Lycopene cleavage products are most likely rapidly metabolized/degraded and their half life should be quite short
Lycopenoid concentrations and half-life • Local tissue production of lycopenoids by CMO-II and other enzymes have the potential to impact metabolic pathways in the way that retinoids do • Harrison (see talk on Monday afternoon) has shown that some β-apo-carotenoids are RAR antagonists
Potential toxicity of lycopenoids • Amengual et al (FASEB J. , 2010) have demonstrated in CMO-II KO mice that excess dietary carotenoids impair respiration, produce excess ROS and causes mitochondrial dysfunction (von Lintig talk on Weds) • As with other nutrients, metabolic effects of carotenoid metabolites may have “U-shaped” curves, especially in persons with SNPs or mutations of key metabolic enzymes or transporters
Overall Conclusions: 1. Cleavage products of lycopene are metabolically active 2. Lycopenoids are produced by CMO-II 3. Lycopene interacts with CMO-II to impact androgen metabolism 4. Lycopenoids may impact lipid metabolism via PPARγ
Acknowledgements • • • NIH grant PHS-1 -RO 1 CA 125384 Dr. Clinton (The Ohio State University) Dr. von Lintig (Case Western Reserve University) Dr. Wyss (DSM Nutritional Products) Erdman Lab members
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