Increased levels of HOCl, a potent oxidant, are typically observed in the plasma and tissues of individuals with inflammatory diseases [37
]. And yet, a functional deficiency of taurine, a potent HOCl scavenger, is a defining feature of diabetes, obesity, depression, hypertension, gout, kidney failure, and autism, among other conditions [38
]. Previous epidemiological evidence has suggested that the intake of lycopene is associated with a reduced risk for many chronic disorders including prostate cancer and heart diseases [40
]. Although most investigators have attributed the potential protective role of lycopene in the prevention of chronic diseases to its antioxidant function [41
], the exact mechanism by which lycopene exerts these antioxidant effects have not been fully elucidated. Using a combination of direct UV-visible, HPLC and LC/APCI/MS, we have shown that increasing concentration of HOCl can alter the availability of lycopene through a mechanism that involves lycopene oxidation and fragmentation. Exposure of lycopene to saturated amounts of HOCl caused a distinct bleaching in color suggesting lycopene destruction. HPLC and LC-MS analysis showed that exposure of lycopene to increasing concentrations of HOCl gave a range of metabolites resulting from the oxidative cleavage of one or more C=C. HOCl may directly mediate the destruction of lycopene by unselective cleavage at any double bond position in a nonenzymatic manner. HOCl-mediated lycopene oxidation was rapid in chloroformic solution, and a total of thirteen different cleavage products have been identified based on their mass signals through the treatment of lycopene solution with a range of HOCl (1:5 to 1:20 molar ratio). The degree of degradation of lycopene (as assessed by the number and chain lengths of the different oxidative metabolites of lycopene) depends mainly on the ratio between HOCl to lycopene, suggesting that multiple molecules of HOCl are consumed per molecule of lycopene. Thus, HOCl interaction with lycopene may serve as a potential mechanism for modulating its availability, and thereby influencing the regulation of local inflammatory and infectious events in vivo
These cleavage products can be classified into three major categories according to the degree of oxidation and their cleavage position within the lycopene molecule: the first group contains lycopene metabolites resulting from single oxidative cleavage. These metabolites contain on one end an aldehyde/or an acid group and on the other end of the metabolite the Ψ-end group of lycopene, and were called apo-lycopenal and apo-lycopenoic acid. Indeed, two apo-lycopenal and two apo-lycopenoic acids were obtained and tentatively identified based on the m/z value. Apo-lycopenal isolated fragments showed that the double bonds 5–6 (or 5′–6′), was affected, whereas apo-lycopenoic acid showed that the double bonds 13–14 (or 13′–14′), 15-15′ and 9–10 (or 9′–10′) were affected by the cleavage. All the apo-lycopenoic acid fragments (acycloretioic acid, apo-14’-lycopenoic acid, and apo-10′-lycopenoic acid) were identified at both low (1:5) and high (1:20) lycopene to HOCl ratio. Apo-lycopenal fragments (apo-6′-lycopenal) were identified at only higher lycopene to HOCl ratio (). The second group resulting from the oxidation and modification of the both end groups of lycopene that could occur at position 1–2 (or 1′–2′) or at any other position to produce lycopene metabolites with: two acids, two aldehydes, or combination of both acid and aldehyde on the two end groups and was called apo-carotendials, because their structure could have been obtained from any carotenoid and not only from lycopene. In case of apocarotendials where the 1–2 (or 1′–2′) in one side and the other side showed that the double bonds (7–8) (or 7′–8′) were affected whereas the corresponding acids showed that the double bonds 7–8 (or 7′–8′), 11–12 (or 11′–12′) and 5–6 (or 5′–16′) were affected by the cleavage. In addition, among the different products isolated, three fragments were oxidatively modified at both ends of lycopene’s hyperconjugated chain. These three products were crocetin semialdehyde, crocetin, and apo-5′, 6′ carotendial (). The third group contains other compounds resulting from further chlorination of lycopene metabolites, called chloro-apo-carotendials. This includes m/z 443 which is a chlorinated lycopene fragment: 2 hydroxy 3 chloro 4,6,8,10,12,14,16,18 docosoctene 1,22 diol 6,11,15,19 tetramethyl. Thus, taken together; the results in our studies suggest that the HOCl-mediated cleavage reaction of lycopene and the degree of oxidation could occur unselectively at any position of the lycopene double bonds independent of HOCl concentration. The formation of long-chains was not detected in higher ratio of lycopene to HOCl (e.g., 1:30, data not shown), suggested that the detected fragments are further oxidized to shorter-chain lengths upon increasing the HOCl concentration.
In the case of potassium permanganate-mediated generation of apo-carotendials, Caris-Veyrat et al.
have shown that only the double bonds 5–6 (or 5′–6′), 7–8 (or 7′–8′ ), 9–10 (or 9′–10′), and 11–12 (or 11′–12′) were affected by the cleavage. But they did not detect apo-carotendials/ones resulting from cleavages of double bonds closer to the center of the molecule (15-15′ C=C) [44
]. No cleavage was detected on the double bond which is the farthest from the center of the molecule, i.e., the 1–2 double bond, which is not conjugated and is trisubstituted. The author attributed this behavior to the non-reactivity of these positions to the oxidation with potassium permanganate [44
]. No products resulting from a double oxidative cleavage (apo-carotendial) were detected upon exposure of lycopene to the oxidative catalytic species trans
-dioxoruthenium( VI)-tetraphenylporphyrin Ru(O)2
(TPP) over a period of 96 h [44
]. Instead (Z)
-isomers, compounds with a molecular weight of m/z
553 = [Mlycopene
+ 16 + H]+
, which we called lycopene monoxides, and cleavage compounds assigned to apo-lycopenals [44
]. Almost all the apo-lycopenals/ones that were obtained by oxidation of lycopene with potassium permanganate were detected, except the short-chain apo-5-lycopenone and apo-7-lycopenal (44). Thus, our results may suggest that HOCl behaves as a stronger oxidant with a higher oxidation potential than permanganate and other oxidants for lycopene destruction.
Many of the metabolites in groups 1and 2 have been reported as their metabolic pathways were investigated extensively while other cleavage products and their functions are either not yet fully explained or not mentioned before in the literature. For example, earlier in vitro
studies by Ben-Aziz et al.
have shown that apo-6′- and apo-8′- lycopene were obtained by oxidation of lycopene with potassium permanganate [42
]. Cross-sequential studies by Ukai et al. described a protocol for the reaction which gave apo-6′-lycopenal as the main product after 44 hours, whereas 50% of lycopene remained intact [43
]. Caris-Veyrat et al. have latter optimized the experimental conditions by modifying the solvent mixture and the phase transfer catalyst to complete oxidized lycopene and obtained mono oxidative cleavage compounds as major products [44
]. After the complete disappearance of lycopene, they detected eight apo-lycopenals giving a range of products from the longest apo-6′-lycopenal to the shortest one detected apo-5-lycopenone, three apo-lycopenones, and six apo-carotendials [45
]. Using HPLC analysis with UV–visible detection Kim et al. have tentatively identified mono cleavage compounds of lycopene [46
]. Exposure of lycopene to atmospheric oxygen and perfusion of ozone has led to tentatively identify a (E, E, E)-4-methyl-8-oxo-2,4,6-nonatrienal [45
]. Kim et al.
isolated and identified a cleavage product of lycopene from an autoxidation mixture of lycopene products [46
Previous studies have also shown that lycopene undergoes oxidative degradation in vivo
]. Some of the lycopene cleavage products identified in this work () have been shown to participate in biological effects in animal models and cell culture system. Indeed, several of lycopene metabolites have been found in human milk and serum [47
]. Consistent with our current finding, these studies have concluded that lycopene can unselectively been cleaved at any double bond position in nonenzymatic manner when organisms are subjected to oxidative stress (33,44,45,48). Since several of the HOCl-mediated lycopene cleavage products of different chain lengths assigned in group 1 and 2 () have been identified and shown to be associated with reduced risk of certain cancers [49
], we believe that our work could be biologically relevant. Indeed, it has been thought that lycopene metabolites may induce changes in the gene level attaining the expression of relevant genes and serve as anticancer agents [53
]. Related studies by Nara et al. have shown that a mixture of oxidation products of lycopene induced apoptosis in HL-60 cells following incubation for 24 h, at 37°C [54
]. Kotake-Nara et al.
have shown that acyclo-retinoic acid, the centrally cleaved metabolite of lycopene, reduces cell viability by inducing apoptosis in human prostate cancer cells [55
]. Aust et al.
, have show that 2,7,11-trimethyl-tetradecahexaene-1,14-dial, generated by lycopene degradation, products in cell signaling enhancing cell-to-cell communication through gap junctions in rat liver epithelial WB-F344 cells [56
]. In addition, apo-10'-lycopenoic acid was also found to be a lycopene metabolite in ferret lung tissue [57
]. In parallel, several other lycopene metabolites with different functional groups have previously been reported, for example, 5,6-dihydroxy-5',6'-dihydrolycopene and 2,6-cyclolycopene-1,5-diol A and B have been found in the extracts of human serum/plasma [47
Identifying similarities and differences in the interactions between HOCl and various oxidants yields valuable mechanistic insights into the potential biochemical and functional significance of HOCl−lycopene interactions both in vitro and in vivo. On the basis of the present results, previous published studies of the interaction of HOCl with carotenoids, and the exposure of lycopene to different oxidants (e.g., permanganates and metalloporphyrins), we have generated a stepwise scheme that shows possible pathways for the generation of different lycopene cleavage products after the exposure to increasing concentration of HOCl (). In this reaction, lycopene interacts with the HOCl molecule in which the chloride atom of HOCl acts as an electrophile and the electron rich olefin initially acts as the nucleophile. When the Cl atom adds across the double bond, it does so in such a mannar in which a pseudo-secondary carbo-cation is formed transforming to a more stable chloronium ion. Addition of the hydroxide proceeds by obeying the regioselectivity of nucleophilic chloronium ion ring opening. The semi-halohydrin then undergoes an epoxide ring forming reaction via an intramolecular SN2 type in which a chlorine atom is displaced. Reaction of the epoxide with a second molecule of HOCl (deprotonated by Cl−) causes cleavage of the terminal carbon-carbon bond and the generation of an aldehyde (which is one carbon shorter than the parent epoxide from which it was formed). Further oxidation of the aldehyde by a third molecule of HOCl leads to the formation of carboxylic acid. Thus, it is evident that one molecule of lycopene (which has thirteen C=C) has the potential capacity to scavenge multiple molecules of HOCl.
Proposed chemical mechanism showing the stepwise oxidation of lycopene by HOCl.
The degree of reproducibility and similarity of the isolates increased progressively when the LC/MS analyses were carried out immediately after the extraction of the cleavage products in chloroformic solutions and subsequently as dry powders. The delay of 20–45 h in LC/MS analysis caused a noticeable decrease in the chlorinated compounds (group 3 ), indicating that the chlorinated molecules are relatively unstable and may decay to non-chlorinated species such as aldehydes and carboxylic acids. The metabolites in groups 3 and their metabolic pathways have not been previously reported. The excessive HOCl generated under inflammatory conditions when phagocytes are activated and MPO is released may play a harmful role due to its ability to react eagerly with a variety of biological molecules, and its ability to chlorinate as well as oxidize biomoleules [59
]. Indeed, there is substantial evidence that MPO and HOCl play a role in atherosclerosis, diabetes and asthma, and HOCl-mediated tissue injury has also been found to result in an increase in inflammatory disorders as determined by increased levels of free and protein-bound chlorotyrosine and α-chloro fatty aldehydes [reviewed in 60
The generation of a series of chlorohydrins of cholesterol[6-β-chloro-cholestane-(3β,5α)-diol, 5-α-chloro-cholestane-(3β,6β)-diol and 6-α-chloro-cholestane-(3β,5β)-diol has been observed following exposure to HOCl, as well as dichlorinated product, 5,6-dichloro-cholestane-3β-ol [62
]. In addition to electrophilic addition, HOCl can utilize N-halogenation reactions at amines in the head groups of phosphatidylethanolamines and phosphatidylserines, yielding corresponding chloramines [64
]. These intermediate products are initial short lived molecules, and tend to decay to a more stable non-chlorinated species such as aldehydes. HOCl reacts very rapidly with the sulfur-containing side chains of methionine and cysteine residues and, at a slightly lower rate constant, with amines and other nitrogen-containing residue [66
]. The ability of lycopene to scavenge HOCl formed during inflammatory situations is more likely to prevent proteins and unsaturated phospholipids from MPO- and HOCl-induced damage. Thus reduced lycopene availability and/or increased consumption as assessed by the degree of its fragmentation and modification may reflect the basic mechanism of increasing the risk of diseases induced by MPO and HOCl.
In summary, inhibition of MPO or removing its downstream final product, HOCl, is an attractive target for preventing HOCl-mediated tissue injury and progression of inflammatory diseases. In this study we advance the current knowledge of lycopene antioxidant properties and show for the first time that lycopene may serve as a potent scavenger of HOCl. The interplay between lycopene and HOCl may have a broad implication in the function of inflammatory biological systems throughout the body. Further evaluation is necessary to assess whether lycopene could represent an interventional approach to minimize the deleterious effects associated with inflammation.