Carotenoids in Health as Studied by Omics-Related Endpoints.

Carotenoids have been associated with risk reduction for several chronic diseases, including the association of their dietary intake/circulating levels with reduced incidence of obesity, type 2 diabetes, certain types of cancer, and even lower total mortality. In addition to some carotenoids constituting vitamin A precursors, they are implicated in potential antioxidant effects and pathways related to inflammation and oxidative stress, including transcription factors such as nuclear factor κB and nuclear factor erythroid 2-related factor 2. Carotenoids and metabolites may also interact with nuclear receptors, mainly retinoic acid receptor/retinoid X receptor and peroxisome proliferator-activated receptors, which play a role in the immune system and cellular differentiation. Therefore, a large number of downstream targets are likely influenced by carotenoids, including but not limited to genes and proteins implicated in oxidative stress and inflammation, antioxidation, and cellular differentiation processes. Furthermore, recent studies also propose an association between carotenoid intake and gut microbiota. While all these endpoints could be individually assessed, a more complete/integrative way to determine a multitude of health-related aspects of carotenoids includes (multi)omics-related techniques, especially transcriptomics, proteomics, lipidomics, and metabolomics, as well as metagenomics, measured in a variety of biospecimens including plasma, urine, stool, white blood cells, or other tissue cellular extracts. In this review, we highlight the use of omics technologies to assess health-related effects of carotenoids in mammalian organisms and models.

Carotenoids and Their Health Benefits as Derived via Their Interactions with Gut Microbiota.

Carotenoids have been related to a number of health benefits. Their dietary intake and circulating levels have been associated with a reduced incidence of obesity, diabetes, certain types of cancer, and even lower total mortality. Their potential interaction with the gut microbiota (GM) has been generally overlooked but may be of relevance, as carotenoids largely bypass absorption in the small intestine and are passed on to the colon, where they appear to be in part degraded into unknown metabolites. These may include apo-carotenoids that may have biological effects because of higher aqueous solubility and higher electrophilicity that could better target transcription factors, i.e., NF-κB, PPARγ, and RAR/RXRs. If absorbed in the colon, they could have both local and systemic effects. Certain microbes that may be supplemented were also reported to produce carotenoids in the colon. Although some bactericidal aspects of carotenoids have been shown in vitro, a few studies have also demonstrated a prebiotic-like effect, resulting in bacterial shifts with health-associated properties. Also, stimulation of IgA could play a role in this respect. Carotenoids may further contribute to mucosal and gut barrier health, such as stabilizing tight junctions. This review highlights potential gut-related health-beneficial effects of carotenoids and emphasizes the current research gaps regarding carotenoid-GM interactions.

Carotenoids in orange carrots mitigate non-alcoholic fatty liver disease progression.

Background: Carotenoids are abundant in colored fruits and vegetables. Non-alcoholic fatty liver disease (NAFLD) is a global burden and risk factor for end-stage hepatic diseases. This study aims to compare the anti-NAFLD efficacy between carotenoid-rich and carotenoid-deficient vegetables. Materials and methods: Male C57BL/6J mice were randomized to one of four experimental diets for 15 weeks (n = 12 animals/group): Low-fat diet (LFD, 10% calories from fat), high-fat diet (HFD, 60% calories from fat), HFD with 20% white carrot powders (HFD + WC), or with 20% orange carrot powders (HFD + OC). Results: We observed that carotenoids in the orange carrots reduced HFD-induced weight gain, better than white carrots. Histological and triglyceride (TG) analyses revealed significantly decreased HFD-induced hepatic lipid deposition and TG content in the HFD + WC group, which was further reduced in the HFD + OC group. Western blot analysis demonstrated inconsistent changes of fatty acid synthesis-related proteins but significantly improved ACOX-1 and CPT-II, indicating that orange carrot carotenoids had the potential to inhibit NAFLD by improving ß-oxidation. Further investigation showed significantly higher mRNA and protein levels of PPARα and its transcription factor activity. Conclusion: Carotenoid-rich foods may display more potent efficacy in mitigating NAFLD than those with low carotenoid levels.

The Role of ß-Carotene in Colonic Inflammation and Intestinal Barrier Integrity.

Background: Carotenoids are naturally occurring pigments accounting for the brilliant colors of fruits and vegetables. They may display antioxidant and anti-inflammatory properties in humans besides being precursors to vitamin A. There is a gap of knowledge in examining their role within colonic epithelial cells. We proposed to address this research gap by examining the effects of a major dietary carotenoid, ß-carotene, in the in vitro epithelial cell model. Methods: We examined the function of ß-carotene in the lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway. We conducted western blotting assays to evaluate expressions of TLR4 and its co-receptor, CD14. We also examined NF-κB p65 subunit protein levels in the model system. Furthermore, we studied the impact of ß-carotene on the tight junction proteins, claudin-1, and occludin. We further carried out immunocytochemistry experiments to detect and visualize claudin-1 expression. Results: ß-Carotene reduced LPS-induced intestinal inflammation in colonic epithelial cells. ß-Carotene also promoted the levels of tight junction proteins, which might lead to enhanced barrier function. Conclusions: ß-Carotene could play a role in modulating the LPS-induced TLR4 signaling pathway and in enhancing tight junction proteins. The findings will shed light on the role of ß-carotene in colonic inflammation and also potentially in metabolic disorders since higher levels of LPS might induce features of metabolic diseases.

In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii.

Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (ß-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled ß-carotene synthesis (194 µg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional ß-carotene recovered in feces was 56-fold higher than the ß-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.

The Promising Effects of Astaxanthin on Lung Diseases.

Astaxanthin (ASX) is a naturally occurring xanthophyll carotenoid. Both in vitro and in vivo studies have shown that it is a potent antioxidant with anti-inflammatory properties. Lung cancer is the leading cause of cancer death worldwide, whereas other lung diseases such as chronic obstructive pulmonary disease, emphysema, and asthma are of high prevalence. In the past decade, mounting evidence has suggested a protective role for ASX against lung diseases. This article reviews the potential role of ASX in protecting against lung diseases, including lung cancer. It also summarizes the underlying molecular mechanisms by which ASX protects against pulmonary diseases, including regulating the nuclear factor erythroid 2-related factor/heme oxygenase-1 pathway, NF-κB signaling, mitogen-activated protein kinase signaling, Janus kinase-signal transducers and activators of transcription-3 signaling, the phosphoinositide 3-kinase/Akt pathway, and modulating immune response. Several future directions are proposed in this review. However, most in vitro and in vivo studies have used ASX at concentrations that are not achievable by humans. Also, no clinical trials have been conducted and/or reported. Thus, preclinical studies with ASX treatment within physiological concentrations as well as human studies are required to examine the health benefits of ASX with respect to lung diseases.

Lycopene Protects against Smoking-Induced Lung Cancer by Inducing Base Excision Repair.

BACKGROUND: Oxidative stress plays a critical role in lung cancer progression. Carotenoids are efficient antioxidants. The objective of this study was to explore the efficacy of all-trans retinoic acid (ATRA) and carotenoids in cigarette smoke-induced oxidative stress within A549 human lung cancer epithelial cells. METHODS: A549 cells were pretreated with 1-nM, 10-nM, 100-nM, 1-µM and 10-µM ATRA, ß-carotene (BC) and lycopene for 24 h, followed by exposure to cigarette smoke using a smoking chamber. RESULTS: The OxyBlot analysis showed that smoking significantly increased oxidative stress, which was inhibited by lycopene at 1 nM and 10 nM (p < 0.05). In the cells exposed to smoke, lycopene increased 8-oxoguanine DNA glycosylase (OGG1) expression at 1 nM, 10 nM, 100 nM, and 1 µM (p < 0.05), but not at 10 µM. Lycopene at lower doses also improved Nei like DNA glycosylases (NEIL1, NEIL2, NEIL3), and connexin-43 (Cx43) protein levels (p < 0.05). Interestingly, lycopene at lower concentrations promoted OGG1 expression within the cells exposed to smoke to an even greater extent than the cells not exposed to smoke (p < 0.01). This may be attributed to the increased SR-B1 mRNA levels with cigarette smoke exposure (p < 0.05). CONCLUSIONS: Lycopene treatment at a lower dosage could inhibit smoke-induced oxidative stress and promote genome stability. These novel findings will shed light on the molecular mechanism of lycopene action against lung cancer.

The Colors of Health: Chemistry, Bioactivity, and Market Demand for Colorful Foods and Natural Food Sources of Colorants.

There is an increasing consumer demand for natural colors in foods. However, there is a limited number of available natural food sources for use by the food industry because of technical and regulatory limitations. Natural colors are less stable and have less vibrant hues compared to their synthetic color counterparts. Natural pigments also have known health benefits that are seldom leveraged by the food industry. Betalains, carotenoids, phycocyanins, and anthocyanins are major food colorants used in the food industry that have documented biological effects, particularly in the prevention and management of chronic diseases such as diabetes, obesity, and cardiovascular disease. The color industry needs new sources of stable, functional, and safe natural food colorants. New opportunities include sourcing new colors from microbial sources and via the use of genetic biotechnology. In all cases, there is an imperative need for toxicological evaluation to pave the way for their regulatory approval.

Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model.

Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity.

Plasma proteome correlates of lipid and lipoprotein: biomarkers of metabolic diversity and inflammation in children of rural Nepal.

Proteins involved in lipoprotein metabolism can modulate cardiovascular health. While often measured to assess adult metabolic diseases, little is known about the proteomes of lipoproteins and their relation to metabolic dysregulation and underlying inflammation in undernourished child populations. The objective of this population study was to globally characterize plasma proteins systemically associated with HDL, LDL, and triglycerides in 500 Nepalese children. Abnormal lipid profiles characterized by elevated plasma triglycerides and low HDL-cholesterol (HDL-C) concentrations were common, especially in children with subclinical inflammation. Among 982 proteins analyzed, the relative abundance of 11, 12, and 52 plasma proteins was correlated with LDL-cholesterol (r = -0.43∼0.70), triglycerides (r = -0.39∼0.53), and HDL-C (r = -0.49∼0.79) concentrations, respectively. These proteins included apolipoproteins and numerous unexpected intracellular and extracellular matrix binding proteins, likely originating in hepatic and peripheral tissues. Relative abundance of two-thirds of the HDL proteome varied with inflammation, with acute phase reactants higher by 4∼40%, and proteins involved in HDL biosynthesis, cholesterol efflux, vitamin transport, angiogenesis, and tissue repair lower by 3∼20%. Untargeted plasma proteomics detects comprehensive sets of both known and novel lipoprotein-associated proteins likely reflecting systemic regulation of lipoprotein metabolism and vascular homeostasis. Inflammation-altered distributions of the HDL proteome may be predisposing undernourished populations to early chronic disease.

Plasma proteins associated with circulating carotenoids in Nepalese school-aged children.

Carotenoids are naturally occurring pigments that function as vitamin A precursors, antioxidants, anti-inflammatory agents or biomarkers of recent vegetable and fruit intake, and are thus important for population health and nutritional assessment. An assay approach that measures proteins could be more technologically feasible than chromatography, thus enabling more frequent carotenoid status assessment. We explored associations between proteomic biomarkers and concentrations of 6 common dietary carotenoids (α-carotene, ß-carotene, lutein/zeaxanthin, ß-cryptoxanthin, and lycopene) in plasma from 500 6-8 year old Nepalese children. Samples were depleted of 6 high-abundance proteins. Plasma proteins were quantified using tandem mass spectrometry and expressed as relative abundance. Linear mixed effects models were used to determine the carotenoid:protein associations, accepting a false discovery rate of q < 0.10. We quantified 982 plasma proteins in >10% of all child samples. Among these, relative abundance of 4 were associated with ß-carotene, 11 with lutein/zeaxanthin and 51 with ß-cryptoxanthin. Carotenoid-associated proteins are notably involved in lipid and vitamin A transport, antioxidant function and anti-inflammatory processes. No protein biomarkers met criteria for association with α-carotene or lycopene. Plasma proteomics may offer an approach to assess functional biomarkers of carotenoid status, intake and biological function for public health application. Original maternal micronutrient trial from which data were derived as a follow-up activity was registered at ClinicalTrials.gov: NCT00115271.

Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors.

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.

Synthesis of apo-13- and apo-15-lycopenoids, cleavage products of lycopene that are retinoic acid antagonists.

Consumption of the tomato carotenoid, lycopene, has been associated with favorable health benefits. Some of lycopene's biological activity may be due to metabolites resulting from cleavage of the lycopene molecule. Because of their structural similarity to the retinoic acid receptor (RAR) antagonist, ß-apo-13-carotenone, the "first half" putative oxidative cleavage products of the symmetrical lycopene have been synthesized. All transformations proceed in moderate to good yield and some with high stereochemical integrity allowing ready access to these otherwise difficult to obtain terpenoids. In particular, the methods described allow ready access to the trans isomers of citral (geranial) and pseudoionone, important flavor and fragrance compounds that are not readily available isomerically pure and are building blocks for many of the longer apolycopenoids. In addition, all of the apo-11, apo-13, and apo-15 lycopenals/lycopenones/lycopenoic acids have been prepared. These compounds have been evaluated for their effect on RAR-induced genes in cultured hepatoma cells and, much like ß-apo-13-carotenone, the comparable apo-13-lycopenone and the apo-15-lycopenal behave as RAR antagonists. Furthermore, molecular modeling studies demonstrate that the apo-13-lycopenone efficiently docked into the ligand binding site of RARα. Finally, isothermal titration calorimetry studies reveal that apo-13-lycopenone acts as an antagonist of RAR by inhibiting coactivator recruitment to the receptor.

Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism.

RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 µM atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.

Carotenoid metabolism in mammals, including man: formation, occurrence, and function of apocarotenoids.

Vitamin A was recognized as an essential nutrient 100 years ago. In the 1930s, it became clear that dietary ß-carotene was cleaved at its central double to yield vitamin A (retinal or ß-apo-15'-carotenal). Thus a great deal of research has focused on the central cleavage of provitamin A carotenoids to form vitamin A (retinoids). The mechanisms of formation and the physiological role(s) of noncentral (eccentric) cleavage of both provitamin A carotenoids and nonprovitamin A carotenoids has been less clear. It is becoming apparent that the apocarotenoids exert unique biological activities themselves. These compounds are found in the diet and thus may be absorbed in the intestine, or they may form from enzymatic or nonenzymatic cleavage of the parent carotenoids. The mechanism of action of apocarotenoids in mammals is not fully worked out. However, as detailed in this review, they have profound effects on gene expression and work, at least in part, through the modulation of ligand-activated nuclear receptors. Understanding the interactions of apocarotenoids with other lipid-binding proteins, chaperones, and metabolizing enzymes will undoubtedly increase our understanding of the biological roles of these carotenoid metabolites.

The formation, occurrence, and function of ß-apocarotenoids: ß-carotene metabolites that may modulate nuclear receptor signaling.

ß-Carotene is the major dietary source of provitamin A. Central cleavage of ß-carotene yields 2 molecules of retinal followed by further oxidation to retinoic acid. Eccentric cleavage of ß-carotene occurs at double bonds other than the central double bond, and the products of these reactions are ß-apocarotenals and ß-apocarotenones. We reviewed recent developments in 3 areas: 1): the enzymatic production of ß-apocarotenoids in higher animals; 2) the occurrence of ß-apocarotenoids in foods and animal tissues; and 3) the biological activity of ß-apocarotenoids, particularly on retinoid receptors. HPLC-mass spectrometry techniques were developed to quantify these compounds in mouse serum and tissues and in foods. ß-Apo-10'- and -12'-carotenals were detected in mouse serum and liver. ß-Apo-8'-, ß-apo-10'-, ß-apo-12'-, and ß-apo-14'-carotenals and ß-apo-13-carotenone were detected in orange-fleshed melons. Transactivation assays were performed to see whether apocarotenoids activate or antagonize retinoid X receptor (RXR) α. Reporter gene constructs and retinoid receptor (RXRα) were transfected into cells, which were used to perform quantitative assays for the activation of this ligand-dependent transcription factor. None of the ß-apocarotenoids significantly activated RXRα. However, ß-apo-13-carotenone antagonized the 9-cis-retinoic acid activation of RXRα. Competitive radioligand binding assays showed that this antagonist competes directly with the agonist for binding to purified receptor, a finding confirmed by molecular modeling studies. These findings suggest that a possible biological function of ß-apocarotenoids is their ability to interfere with nuclear receptor signaling. Recent work showed that ß-apo-13-carotenone is also a high-affinity antagonist of all 3 retinoic acid receptors (RARα, RARß, and RARγ).

Naturally occurring eccentric cleavage products of provitamin A ß-carotene function as antagonists of retinoic acid receptors.

ß-Carotene is the major dietary source of provitamin A. Central cleavage of ß-carotene catalyzed by ß-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA), which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of ß-carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are ß-apocarotenals and ß-apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the ß-apocarotenoids significantly activated RARs. Importantly, however, ß-apo-14'-carotenal, ß-apo-14'-carotenoic acid, and ß-apo-13-carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that ß-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. ß-Apo-13-carotenone and the ß-apo-14'-carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3-5 nm ß-apo-13-carotenone was present in human plasma. These findings suggest that ß-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary ß-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.

The eccentric cleavage product of ß-carotene, ß-apo-13-carotenone, functions as an antagonist of RXRα.

In this study, we investigated the effects of eccentric cleavage products of ß-carotene, i.e. ß-apocarotenoids (BACs), on retinoid X receptor alpha (RXRα) signaling. Transactivation assays were performed to test whether BACs activate or antagonize RXRα. Reporter gene constructs (RXRE-Luc, pRL-tk) and RXRα were transfected into Cos-1 cells and used to perform these assays. None of the BACs tested activated RXRα. Among the compounds tested, ß-apo-13-carotenone was found to antagonize the activation of RXRα by 9-cis-retinoic acid and was effective at concentrations as low as 1 nM. Molecular modeling studies revealed that ß-apo-13-carotenone makes molecular interactions like an antagonist of RXRα. The results suggest a possible function of BACs on RXRα signaling.