Previous studies have shown that ␤-carotene 15,15-monooxygenase catalyzes the cleavage of ␤-carotene at the central carbon 15,15-double bond but cleaves lycopene with much lower activity. However, expressing the mouse carotene 9,10-monooxygenase (CMO2) in ␤-carotene/lycopene-synthesizing and -accumulating Escherichia coli strains leads to both a color shift and formation of apo-10-carotenoids, suggesting the oxidative cleavage of both carotenoids at their 9,10-double bond. Here we provide information on the biochemical characterization of CMO2 of the ferret, a model for human carotenoid metabolism, in terms of the kinetic analysis of ␤-carotene/lycopene cleavage into ␤-apo-10-carotenal/apo-10-lycopenal in vitro and the formation of apo-10-lycopenoids in ferrets in vivo. We demonstrate that the recombinant ferret CMO2 catalyzes the excentric cleavage of both all-trans-␤-carotene and the 5-cis-and 13-cis-isomers of lycopene at the 9,10-double bond but not all-trans-lycopene. The cleavage activity of ferret CMO2 was higher toward lycopene cis-isomers as compared with ␤-carotene as substrate. Iron was an essential co-factor for the reaction. Furthermore, all-trans-lycopene supplementation in ferrets resulted in significant accumulation of cis-isomers of lycopene and the formation of apo-10-lycopenol, as well as upregulation of the CMO2 expression in lung tissues. In addition, in vitro incubation of apo-10-lycopenal with the post-nuclear fraction of hepatic homogenates of ferrets resulted in the production of both apo-10-lycopenoic acid and apo-10-lycopenol, respectively, depending upon the presence of NAD ؉ or NADH as cofactors. Our finding of bioconversion of cis-isomers of lycopene into apo-10-lycopenoids by CMO2 is significant because cis-isomers of lycopene are a predominant form of lycopene in mammalian tissues and apo-lycopenoids may have specific biological activities related to human health.Carotenoids are lipophilic plant pigments with polyisoprenoid structures, typically containing a series of conjugated double bonds in the central chain of the molecule, which makes them susceptible to oxidative cleavage, and isomerization from trans to cis forms, with the formation of potentially bioactive metabolites. Knowledge of the biological effects of carotenoids, particularly for the impact of oxidation on these carotenoids and the potential for beneficial effects of small quantities or harmful effects of large quantities of the resulting metabolic products, has been reviewed recently (1). For provitamin A carotenoids, such as ␤-carotene, ␣-carotene, and ␤-cryptoxanthin, central cleavage is a major pathway leading to vitamin A formation (2, 3). This pathway has been substantiated by the cloning of the central cleavage enzyme at their 15,15Ј-double bond, ␤-carotene 15,15Ј-monooxygenase (CMO1, 2 formerly called ␤-carotene 15,15Ј-dioxygenase) in different species (4, 5), further classification of this enzyme as a non-heme iron monooxygenase (6), and recent biochemical and structural characterizations (7-11). Very rece...

Reactive oxygen species are potentially damaging molecules. An important function of antioxidants is to intercept harmful triplet states, in order to prevent the formation of singlet oxygen, or to quench singlet oxygen directly. However, antioxidants are also reactive towards other active oxygen species such as the hydroxyl radical, the superoxide anion and the non-excited oxygen ground state in the presence of radical initiators. It is well known that¯avonoids and carotenoids show strong antioxidant properties. Polyenes and carotenoids are the best known among the compounds that quench singlet oxygen by ef®cient energy transfer. A large number of modi®ed, synthetic analogues and derivatives have been synthesised to prepare even better quenchers than the natural carotenoids. Phenols are also excellent chain-breaking antioxidants. Recently, many indigoid dyes (including bacterial indigoids) were studied, with the remarkable result that most, but not all, members of this class of chromophores quench singlet oxygen at the diffusion limit and some of them are excellent radical traps. It has been shown in this study that a quantitative assessment of antioxidant properties of avonoids, carotenoids, phenols and natural indigoids can be achieved using the following three assays: (1) oxygen pressure dependence; (2) peroxide formation; (3) singlet oxygen quenching. Reactivities towards both excited states and ground state radicals can be properly described by these assays. The remarkable role of b-carotene as an`unusual antioxidant' (Burton GW and Ingold KU, Science 224: 569±573 (1984)) in reactions using various oxygen pressures becomes clearer. The socalled`pro-oxidant effects' concern primarily the antioxidant itself and its degradation, since no or very little damage to the substrate occurs in this type of experiment. Three main categories of antioxidants may be classi®ed: (1) excellent antioxidants that perfectly quench excited states as well as ground state radicals (eg actinioerythrol, astaxanthin); (2) good antioxidants that strongly inhibit peroxide formation but are less ef®cient in quenching excited states (eg¯avonols, tocopherols) or lead to considerable degradation of the antioxidant itself (eg b-carotene, lycopene); (3) moderate antioxidants that fail to excel in both reactivities (eg z-carotene,¯avone).

b -apo-carotenals as well as apo-lycopenals into retinal, (3R)-3-hydroxy-retinal and acycloretinal respectively. In addition, the alcohols of these substrates were cleaved to yield the corresponding retinal derivatives.

High intake of lycopene has been associated with a lower risk of a variety of cancers including lung cancer. We recently showed that lycopene can be converted to apo-10'-lycopenoids [Hu et al. (2006). J. Biol. Chem., 281, 19327-19338] in mammalian tissues both in vitro and in vivo, raising the question of whether apo-10'-lycopenoids have biological activities against lung carcinogenesis. In the present study, we report that apo-10'-lycopenoic acid inhibited the growth of NHBE normal human bronchial epithelial cells, BEAS-2B-immortalized normal bronchial epithelial cells and A549 non-small cell lung cancer cells. This inhibitory effect of apo-10'-lycopenoic acid was associated with decreased cyclin E, inhibition of cell cycle progression from G(1) to S phase and increased cell cycle regulators p21 and p27 protein levels. In addition, apo-10'-lycopenoic acid transactivated the retinoic acid receptor beta (RARbeta) promoter and induced the expression of RARbeta. We further examined the effect of apo-10'-lycopenoic acid treatment on 4-(N-methyl-N-nitrosamino)-1-(3-pyridal)-1-butanone (NNK)-induced lung tumorigenesis in the A/J mouse model. We found that the lung tumor multiplicity was decreased dose dependently from an average of 16 tumors per mouse in the NNK injection alone group, to an average of 10, 7 and 5 tumors per mouse in groups injected with NNK and supplemented with 10, 40 and 120 mg/kg diet of apo-10'-lycopenoic acid, respectively. These observations demonstrate that apo-10'-lycopenoic acid is a biological active metabolite of lycopene and suggest that apo-10'-lycopenoic acid is a potential chemopreventive agent against lung tumorigenesis.

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Xanthophyll carotenoids, such as lutein, zeaxanthin and β-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,15'-monooxygenase (CMO1) has been shown to be involved in vitamin A formation, while recent studies suggest that carotene-9',10'-monooxygenase (CMO2) may have a broader substrate specificity than previously recognized. In this in vitro study, we investigated baculovirusgenerated recombinant ferret CMO2 cleavage activity towards the carotenoid substrates zeaxanthin, lutein and β-cryptoxanthin. Utilizing HPLC, LC-MS and GC-MS, we identified both volatile and non-volatile apocarotenoid products including 3-OH-β-ionone, 3-OH-α-ionone, β-ionone, 3-OH-α-apo-10'-carotenal, 3-OH-β-apo-10'-carotenal, and β-apo-10'-carotenal, indicating cleavage at both the 9,10 and 9',10' carbon-carbon double bond. Enzyme kinetic analysis indicated the xanthophylls zeaxanthin and lutein are preferentially cleaved over β-cryptoxanthin, indicating a key role of CMO2 in non-provitamin A carotenoid metabolism. Furthermore, incubation of 3-OH-β-apo-10'-carotenal with CMO2 lysate resulted in the formation of 3-OH-β-ionone. In the presence of NAD + , in vitro incubation of 3-OH-β-apo-10'-carotenal with ferret hepatic homogenates formed 3-OH-β-apo-10'-carotenoic acid. Since apo-carotenoids serve as important signaling molecules in a variety of biological processes, enzymatic cleavage of xanthophylls by mammalian CMO2 represents a new avenue of research regarding vertebrate carotenoid metabolism and biological function.

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Lycopene has been shown to be beneficial in protecting against high-fat diet-induced fatty liver. The recent demonstration that lycopene can be converted by carotene 9',10'-oxygenase into a biologically active metabolite, ALA, led us to propose that the function of lycopene can be mediated by ALA. In the present study, male ob/ob mice were fed a liquid high-fat diet (60% energy from fat) with ALA supplementation (ALA group, 240 μg · kg body weight(-1) · d(-1)) or without ALA supplementation as the control (C group) for 16 wk. Steatosis, SIRT1 expression and activity, genes involved in lipid metabolism, and ALA concentrations in the livers of mice were examined. The results showed that ALA supplementation resulted in a significant accumulation of ALA in the liver and markedly decreased the steatosis in the ALA group without altering body and liver weights compared to the C group. The mRNA and protein levels of hepatic SIRT1 were higher in the ALA group compared to the C group. SIRT1 activity also was higher in the ALA group, as indicated by the lower levels of acetylated forkhead box class O1 protein levels. In addition, the mRNA level of acetyl CoA carboxylase 1 was significantly lower in the ALA group than in the C group. Because SIRT1 plays a key role in lipid homeostasis, the present study suggests that the lycopene metabolite, ALA, protects against the development of steatosis in ob/ob mice by upregulating SIRT1 gene expression and activity.