on-line at: www.actabp.pl Are lycopene metabolites metabolically active?*

Lycopene is the most abundant carotenoid found in tomatoes and thus has been touted as the bioactive component for the reduced risk of chronic diseases such as prostate cancer. We and others hypothesize that lycopene metabolites are responsible for positively modulating biomarkers and risk factors for the prevention of chronic diseases. Lycopene metabolites circulate in serum and accumulate in tissues at concentrations equivalent to bioactive retinoids. Recent studies report that lycopene metabolites reduce the proliferation of cancer cells, induce apoptosis, enhance gap junction communication between cells, alter normal cell cycle progression, and modulate androgen signaling pathways. Here we review recent literature and provide new evidence to suggest that lycopene metabolites may be bioactive at physiological concentrations.


INTRODUCTION
Epidemiological evidence suggests that consumption of lycopene, the red pigment of tomato products and a major carotenoid in human plasma and tissues, is inversely associated with the risk for a number of pathologies including most notably prostate cancer (Giovannucci et al., 1995;Canene-Adams et al., 2005).Based upon recent advances in understanding carotenoid metabolism, we hypothesize that lycopene metabolites may be responsible, at least in part, for this association.
In this review, we will show that lycopene metabolites circulate in serum and accumulate in tissues at physiologically-relevant concentrations in comparison to retinoids, establish that lycopene metabolites are biologically active, and introduce new findings that suggest these metabolites alter serum testosterone levels which are an important risk factor for prostate cancer.

LYCOPENE METABOLITES
Lycopene metabolites, also termed lycopenoids, are poly-isoprenoid compounds of less than 40 carbons in length derived from the parent compound, lycopene (Lindshield et al., 2007).Previous studies identified and characterized lycopenoids and oxidation products generated in vivo; some of these metabolites demonstrate anticancer activity by inhibiting proliferation, inducing apoptosis, or enhancing cell to cell communication (King et al., 1997;Zhang et al., 2003;Ford et al., 2011a).
A series of apo-lycopenals and short-chain carbonyl compounds were produced by in vitro autoxidation of lycopene including apo-14′-lycopenal, apo-12′-lycopenal, apo-10′-lycopenal and apo-6′-lycopenal (Kim et al., 2001).Additional studies have identified other apo-lycopenals in vitro (Khachik et al., 1998a;Khachik et al., 1998b;Caris-Veyrat et al., 2003).Although there are problems associated with the use of in vitro models for this type of work, the chemicals formed in vitro may be important representatives of carotenoid cleavage in the lungs of smokers, tumors in cancer patients, or other oxidatively stressed conditions (Ford et al., 2011a).
Metabolites of lycopene have also been identified in vivo and interestingly, concentrations of lycopenoids are of comparable biological concentrations to retinoids produced from β-carotene (Table 1).Since all trans retinoic and 9-cis retinoic acids are important ligands for a variety of nuclear receptors, it is certainly plausible that some lycopenoids that have similar structure, polarity and molecular weights may be antagonists or agonists to some receptors.
Recent studies from our lab and others have demonstrated that dietary lycopene alters the mRNA expression of CMO-II in animal models.We measured the relative mRNA expression of CMO-II by qRT-PCR in healthy prostate tissue and prostatic tumor from male Copenha-gen rats in response to a 10% tomato powder diet or a control AIN-93G diet (Fig. 1).Quantification of CMO-II mRNA expression was described previously (Ford et al., 2011b), the data was analyzed by student's t-test, and p<0.05 was considered statistically significant.The tomato powder diet significantly reduced the expression of CMO-II in healthy prostate tissue.However, in prostatic tumor tissue, the tomato powder diet induced mRNA expression of CMO-II relative to tissues of rats that were fed the control diet.In addition, we demonstrated that a lycopene-enriched diet significantly reduced the expression of CMO-II in renal tissue of F344 rats which we hypothesize was the result of feedback inhibition by lycopene or its metabolites (Zaripheh et al., 2006).In a study from another laboratory, dietary lycopene increased the expression of CMO-II mRNA by 4-fold in the lungs of ferrets (Hu et al., 2006).The apparent contradictions in these data sets suggest to us that these are tissue specific metabolic and/or oxidative environments that affect lycopene catabolism and/or the possible differential need for lycopene metabolites by specific tissues.We hypothesize that highly oxidative conditions such as lung and tumor tissue may produce or require greater concentrations of lycopene metabolites and therefore inducing CMO-II expression in these tissues.Overall, the effect of carotenoid consumption on the expression of carotenoid cleavage enzymes warrants further investigation.

BIOLOGICAL ACTIVITY OF APO-LYCOPENALS AND APO-LYCOPENOIC ACIDS
Recent strong scientific interest in lycopene metabolism has produced some early evidence to suggest that lycopenoids are biologically active.We recently reported that apo-10′-lycopenal and apo-12′-lycopenal treatment reduced DU145 prostate cancer cell proliferation in a dose-dependent manner in part through regulation of the normal cell cycle (Ford et al., 2011a).A separate group demonstrated that apo-10′-lycopenoic acid reduced proliferation of lung cancer cells and tumorigenesis of an in vivo lung tumor mouse model through Nrf2-mediated induction of phase II detoxifying/antioxidant enzymes (Lian et al., 2007).Future studies will further define the biological benefits of lycopenoids.

BIOLOGICAL ACTIVITY OF LYCOPENE OXIDATION PRODUCTS
Products produced from lycopene through a variety of oxidative procedures have been reported to modulate biological activity.It has been demonstrated that oxidation products of lycopene inhibit the growth of human leukemia cells in vitro (Nara et al., 2001).Specifically, 6 μM of lycopene failed to alter leukemia cell proliferation while 6 μM of oxidative lycopene products (produced from incubation with toluene for 24 hours) dramatically inhibited cell growth by 97% relative to control after 120 hours in culture.In human promyelocytic leukemia cells, a different oxidative product, (E,E,E)-4-methyl-8-oxo-2,4-6-nonatrienal (5-15 μM), also reduced proliferation and induced the essential physiological mechanism to remove damaged DNA by apoptosis (Zhang et al., 2003).In contrast, lycopene treatment alone did not affect apoptosis and had little effect on proliferation rates.Other oxidative products of lycopene have also been shown to inhibit prostate cancer cell growth through induction of apoptosis (Kotake-Nara et al., 2002).In another study, a solution of undefined lycopene oxidative products stimu-  CMO-II expression was analyzed by qRT-PCR using SYBR green fluorescence as described previously (Ford et al., 2011b) and compared to published literature (Wyss 2004;Lindqvist et al., 2005).(Ford et al., 2011b).Bioactive lycopene metabolites lated gap junction communication which is essential to reduce overgrowth of cells as typically found in cancerous tissues (Aust et al., 2003).Another lab demonstrated that an ethanol extract of oxidized lycopene activated the electrophile/antioxidant response element which is known to induce phase II detoxification enzymes in human mammary cancer cells (Linnewiel et al., 2009).
Early studies are starting to suggest that lycopene metabolites play a role in preventing or moderating certain cancer types.Therefore, identification of bioactive lycopene metabolites and further characterization of their in vivo function(s) is critical for cancer research.

PROSTATE CANCER, TESTOSTERONE AND LYCOPENE METABOLITES
Prostate cancer risk is positively associated with induced androgen signaling (Gann et al., 1996;Shaneyfelt et al., 2000).It was first reported in 1941 that the reduction of testosterone levels is a potent therapeutic agent for patients with advanced prostate cancer (Huggins et al., 2002).Moreover, a causal relationship was suggested by a few case reports of prostate cancer patients who previously used androgens as anabolic agents or for medical treatment (Roberts et al., 1986;Ebling et al., 1997).
Recently, we established that testosterone levels in our mouse models are dependent upon an interaction of the expression of carotenoid cleavage enzymes and dietary levels of lycopene.Specifically, we reported that tomato powder or lycopene-containing diets reduced serum and testicular testosterone in CMO-I knock out (CMO-I KO) mice (Ford et al., 2011b).It should be noted that the primary site of whole body testosterone production is from the testis tissue and we demonstrated the testis tissue of CMO-I KO mice have significantly elevated expression of CMO-II.Therefore, it is plausible that CMO-I KO mice have elevated production of lycopene metabolites in testis tissue which effectively reduced serum and testicular testosterone concentrations.Here, we further demonstrate in this mouse model that the expression of key testosterone metabolism genes or receptors is significantly reduced in response to a diet containing 248 nmol lycopene per gram of diet (Fig. 2).The mRNA expression of testicular 5-alpha reductase I, testicular 5-alpha reductase II, and prostatic androgen receptor was measured by qRT-PCR using the SYBR green dye as previously described (Ford et al., 2010).The conversion of testosterone to its more potent androgen dihydrotestosterone, DHT, is catalyzed by the 5-alpha reductase enzyme.Either testosterone or DHT may bind the androgen receptor thus inducing androgen responsive genes.The significant reduction in the expression of these genes by a lycopene-containing diet suggests a reduction in androgen signaling in the testes and prostate.Therefore, it is conceivable that the enhanced production of lycopenoids in the testis of CMO-I KO mice reduced androgen signaling and therefore may potentially reduce prostate cancer risk.

CONCLUSIONS
Lycopene metabolites have been identified in vivo in similar concentrations to the biologically active retinoids.Recent reports suggest that lycopenoids reduce proliferation of cancer cells, induce apoptosis, regulate flow through the cell cycle, induce nuclear transcription factors, enhance cell to cell communication, and reduce androgen signaling.The consumption of lycopene containing foods and the tissue specific expression of carotenoid cleavage enzymes determines tissue lycopenoid concentrations.New reports suggest that these lycopenoids are biologically active and may reduce the risk for chronic diseases.We provide data that suggest that lycopeniods influence androgen metabolism in rodent models.(Ford et al., 2011b).mRNA expression is expressed relative to the ribosomal gene, 18S.mRNA expression of target genes in each tissue was analyzed by student's t-test; (*) p<0.05 considered statistically significant.

Figure 2 .
Figure 2. Relative mRNA expression of steroid pathway genes.Testicular 5 alpha reductase I, testicular 5 alpha reductase II, and prostatic androgen receptor are differentially expressed in CMO-I knock-out mouse tissues in response to a lycopene-containing diet (248 nmol/g diet).CMO-I knock-out mice have significantly elevated expression of testicular CMO-II(Ford et al., 2011b).mRNA expression is expressed relative to the ribosomal gene, 18S.mRNA expression of target genes in each tissue was analyzed by student's t-test; (*) p<0.05 considered statistically significant.

Table 1 . Tissue and serum concentrations of lycopene and β-carotene metabolites.
Prostate and prostatic tumor tissue express CMO-II but expression is differentially affected by consumption of a 10% tomato powder diet containing approximately 13 nmol lycopene per gram of diet.Quantitative RT-PCR analysis of the carotenoid monooxyge- Leenheer et al., 1982;Gajic et al., 2006;Hu et al., 2006;Kopec et al., 2010)10)Figure1.Relative CMO-II mRNA expression.nase II (CMO-II) was carried out in healthy rat prostatic tissue and prostatic tumor tissue from Dunning R3327-H prostate adenocarcinoma orthotopic transplant in male Copenhagen rats.mRNA expression is expressed relative to the ribosomal gene, 18S.Expression of CMO-II in each tissue was analyzed by student's t-test; (*) p<0.05 considered statistically significant.