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Carotenoids, inflammation, and oxidative stress—Implications of cellular signaling pathways and relation to chronic disease prevention

Authors:
  • Luxembourg Institute of Science and Technology-LIST
Carotenoids, inflammation, and oxidative
stressimplications of cellular signaling pathways
and relation to chronic disease prevention
Anouk Kaulmann, Torsten Bohn
Centre de Recherche PublicGabriel Lippmann, Environment and Agro-biotechnologies Department, L-4422 Belvaux, Luxembourg
ARTICLE INFO ABSTRACT
Article history:
Received 19 December 2013
Revised 24 June 2014
Accepted 14 July 2014
Several epidemiologic studies have shown that diets rich in fruits and vegetables reduce the risk
of developing several chronic diseases, such as type 2 diabetes, atherosclerosis, and cancer. These
diseases are linked with systemic, low-grade chronic inflammation. Although controversy
persists on the bioactive ingredients, several secondary plant metabolites have been associated
with these beneficial health effects. Carotenoids represent the most abundant lipid-soluble
phytochemicals, and in vitro and in vivo studies have suggested that they have antioxidant,
antiapoptotic, and anti-inflammatory properties. Recently, many of these properties have been
linked to the effect of carotenoids on intracellular signaling cascades, thereby influencing gene
expression and protein translation. By blocking the translocation of nuclear factor κBtothe
nucleus, carotenoids are able to interact with the nuclear factor κB pathway and thus inhibit the
downstream production of inflammatory cytokines, such as interleukin-8 or prostaglandin E2.
Carotenoids can also block oxidative stress by interacting with the nuclear factor erythroid 2
related factor 2 pathway, enhancing its translocation into the nucleus, and activating phase II
enzymes and antioxidants, such as glutathione-S-transferases. In this review, which is orga nized
into in vitro, animal, and human investigations, we summarized current knowledge on
carotenoids and metabolites with respect to their ability to modulate inflammatory and
oxidative stress pathways and discuss potential dose-health relations. Although many pathways
involved in the bioactivity of carotenoids have been revealed, future research should be directed
toward dose-response relations of carotenoids, theirmetabolites, and their effect on transcription
factors and metabolism.
© 2014 Elsevier Inc. All rights reserved.
Keywords:
Carotenes
Xanthophylls
NF-κB
Nrf2
Transcription factors
Chronic diseases
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Abbreviations: AMD, age-related macular degeneration; ARE, antioxidant response element; BCDO2, β-carotene dioxygenase 2; BCO1,
β-carotene 15,15-oxygenase 1; CAT, catalase; CCL2, chemokine (C-C motif) ligand 2; COX-2, cyclooxygenase 2; CVD, cardiovascular
disease; CXCL, chemokine (C-X-C motif) ligand 2; DMSO, dimethyl sulfoxide; GCL, glutamate cysteine ligase; GPx, glutathione peroxidase;
GSH, glutathione; GSTs, glutathione-S-transferase; HMGB1, high-mobility group box 1; HO-1, heme oxygenase; ICAM-1, intracellular
adhesion molecule 1; IGFBP3, insulin-like growth factor binding protein 3; IKK, IκB kinase; iNOS, nitric oxide synthase; Keap1, kelch-like
ECH-associated protein 1; LDL, low-density lipoprotein; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1,
monocyte chemotactic protein 1; MDA, malondialdehyde; NAD(P)H, nicotinamide adenine dinucleotide phosphate; NEMO, NF-κB
essential modulator; NF-κB, nuclear factor κB; NO, nitric oxide; NOX-2/4, NAD(P)H oxidase; Nrf2, nuclear factor erythroid 2related factor 2;
NQO1, NAD(P)H quinone oxidoreductase 1; 8-OHdG, 8-oxo-2deoxyguanosine; PBMC, peripheral blood mononuclear cell; PGE2,
prostaglandin E2; PGF2α, prostaglandin F2α; RA, retinoic acid; RAR, retinoic acid receptor; ROS, reactive oxygen species; SOD, superoxide
dismutase; STAT, signal transducers and activators of transcription; THF, tetrahydrofuran; TNF-α, tumor necrosis factor α; VCAM-1,
vascular cell adhesion protein 1.
Corresponding author. Centre de Recherche Public - Gabriel Lippmann, 41, rue du Brill, L-4422 Belvaux, Luxembourg. Tel.: +352 470 261
480; fax: +352 470 264.
E-mail address: bohn@lippmann.lu (T. Bohn).
http://dx.doi.org/10.1016/j.nutres.2014.07.010
0271-5317/© 2014 Elsevier Inc. All rights reserved.
Available online at www.sciencedirect.com
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Please cite this article as: Kaulmann A, Bohn T, Carotenoids, inflammation, and oxidative stressimplications of cellular
signaling pathways and relation to chronic disease preven..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.07.010
1. Introduction: carotenoids as antioxidants
Many nutrition and health organizations recommend regular
consumption of fruits and vegetables because it is supposed to
decrease the incidence of several chronic diseases such as
type 2 diabetes [1,2], cardiovascular diseases (CVDs) [3] such as
atherosclerosis [4], and several types of cancer [57]. These
chronic diseases are associated with a systemic, low-grade
chronic inflammatory component that is characterized by
elevated circulating inflammatory markers such as cytokines
(eg, interleukin [IL]-8, IL-6, IL-1, IL-12) [810]; other inflamma-
tory stimulating compounds such as prostaglandin E2 (PGE2)
[11], tumor necrosis factor α(TNF-α)[10], and interferons [10];
acute-phase proteins such as C-reactive protein [12,13];
immune cells associated with inflammatory responses such
as macrophages [14] or eosinophiles [15];andelevated
markers of oxidative stress, for example, prostaglandins [16],
isoprostanes [17], oxidized cholesterol [18], or oxidized lipid
compounds such as malondialdehyde (MDA) [19].These
factors may result in additional tissue damage [20] and
eventually aggravate disease.
Despite other potential dietary confounding factors such as
vitamin C, vitamin E, or dietary fiber, several studies have
attributed observed beneficial health effects to the consump-
tion of secondary plant compounds such as polyphenols
[21,22] and carotenoids [23,24]. Among those, carotenoids
reach the highest plasma and tissue concentrations, ca. 2 μM
[25], despite their lower intake compared with, for example,
polyphenols [26]. The most abundant carotenoids in plasma
include lycopene, β-carotene, and lutein [25]. In addition, their
plasma half-life is relatively long (days to weeks compared
with 2-30 hours for polyphenols) because of their fat solubility,
limited phase II metabolism, and decreased renal clearance
[2729]. Carotenoid consumption and tissue levels have been
related to the prevention of cancer [30,31], diabetes [1,23,32],
and inflammatory bowel diseases [33,34].
Carotenoids are liposoluble C-40based isoprenoid pig-
ments. They are characterized by an extended conjugated
π-electron system that can only be synthesized by plants and
microorganisms [35]. Animals, including humans, must rely
on dietary uptake. To date, approximately 700 different
carotenoid species have been identified, but only 50 have
been reported to play a role in the human diet [36], with an
intake of ca. 5-15 mg/d per capita [37]. Carotenoids can be
separated into the oxygen-devoid carotenes and the oxygen-
containing xanthophylls [3]. They can be further classified into
provitamin A carotenoids (eg, β-carotene and β-cryptoxanthin)
and the nonprovitamin A carotenoids, which cannot be
converted to retinal (eg, lycopene and lutein) [38].
The extended π-electron system is an important feature of
carotenoids because it aids in stabilizing unpaired electrons
after radical quenching [39]. Because of this conjugated
double-bond structure, carotenoids are strong scavengers of
singlet oxygen (
1
O
2
) and peroxyl radicals [40]. They either act
via physical quenching, electron acceptance, or donation [41]
or via hydrogen abstraction/acceptance [42]. Singlet oxygen
scavenging by carotenoids depends largely on physical
quenching, that is, a direct energy transfer between the 2
molecules. This scavenging depends on the number of
conjugated double bonds [43]. Thus, carotenoids with more
extended π-electron systems, such as lycopene, are generally
reported to constitute stronger antioxidants compared with
phytoene/phytofluene [44].
The carotenoids also play an important role in their
orientation within biological membranes [45]. As lipid-soluble
molecules, different carotenoid structures are found in
lipophilic environments and lipid/water interfaces. Xantho-
phylls, which are less hydrophobic than carotenes, are found
in cellular membranes at the lipid/aqueous interface, and they
can scavenge lipid and aqueous phase radicals [41]. Carotenes
scavenge radicals in the lipid phase, as they are mostly located
deep in the apolar core of lipid membranes [46]. Thus, within
cells, carotenoids are affiliated with various types of mem-
branes, such as the outer cell membrane, but also the
mitochondria and the nucleus [47]. They also can be found
in liposomes [48], whereas their free occurrence in the cytosol
is rather low [47]. As a consequence, carotenoids play an
important role in protecting cellular membranes [49] and
lipoproteins [50] against damage by peroxyl radicals.
In addition to their scavenging function toward several
reactive oxygen species (ROS), there is growing awareness that
carotenoids may also act via more indirect pathways. This
indirect route may include interactions with cellular signaling
cascades, such as nuclear factor κB (NF-κB), mitogen-activated
protein kinase (MAPK), or nuclear factor erythroid 2related
factor 2 (Nrf2) [51,52]. Because of their rather low tissue
concentrations and the regulation of antioxidant balance by
numerous other endogenous compounds, the capacity for
scavenging radicals is of biological relevance. After their
cellular uptake from mixed micelles and the resulting
symmetric (β-carotene-oxygenase 1, or BCO1) and assymetric
apocarotenoids (β-carotene-di-oxygenase 2, BCDO2, their
activity depending on genetic factors and the administered
dosage [53]), appear to be bioactive.
Presenting the findings from in vitro, animal, and human
investigations, this review aims to summarize current knowl-
edge on the part carotenoids may play in inhibiting inflam-
matory and oxidative stress related processes by interacting
with cellular signaling cascades. Search criteria in PubMed
included the terms carotenoidscombined with one of the
following: inflammation,”“oxidative stress,”“meta-
analyses,”“NF-κB,”“reactive oxygen species,”“MAPK,
animal studies,or Nrf2; the results were then further
filtered manually. The literature was searched from inception
of PubMed until the present (2014). The initial search
yielded 1657 potential studies, with 215 of these selected for
this review.
2. Oxidative stress, inflammation, and intracellular
signaling cascades
Inflammation, under normal conditions, is a protective
mechanism of tissues against endogenous and exogenous
damage [54]. Several agents and conditions that could lead to
inflammation are known, such as microbial and viral infec-
tions, autoimmune diseases, exposure to allergens or toxic
chemicals, and even metabolic disturbances that include
2NUTRITION RESEARCH XX (2014) XXXXXX
Please cite this article as: Kaulmann A, Bohn T, Carotenoids, inflammation, and oxidative stressimplications of cellular
signaling pathways and relation to chronic disease preven..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.07.010
obesity [55]. Two stages of inflammation are distinguished,
the acute and chronic phases. Acute inflammation is the
initial stage that persists for only a short time, and it is
normally beneficial to the host because it helps to reestablish
normal homeostasis, for example by digesting foreign bacte-
ria. However, if persisting over a prolonged period, this state is
referred to as chronic inflammation. This is harmful for the body
because it can result in abnormal physiological responses,
increase the risk of cellular damage, and lead to the
development of chronic diseases, such as cancer [55].
During inflammation, cells of the immune system such as
macrophages and leucocytes are recruited to the site of
damage. This results in a respiratory burst,an overproduc-
tion of ROS, and leads to oxidative stress and damage of
important biomolecules, such as proteins or DNA [54]. The
inflamed cells also produce soluble mediators such as
cytokines, chemokines, and metabolites of arachidonic acid
(ie, prostaglandins), which further recruit macrophages and
are important key activators of different signal transduction
cascades and transcription factors, such as NF-κBor
Nrf2 (Figure). The activation of these cascades and transcrip-
tion factors then results in the production and secretion of
cellular stress responses such as cyclooxygenase 2 (COX-2),
inducible nitric oxide synthase (iNOS), chemokines, and cyto-
kines [55]. Transcription factors, specifically NF-κBandNrf2,
have been associated with inflammation and oxidative stress
responses, respectively.
Nuclear factor κB is responsible for the transcription of a
variety of genes that regulate inflammatory responses. When
cells are not stimulated, NF-κB is bound to its inhibitory
Receptor
(IKK) inactive
Activated IKK
Iκ-B
NF-κB
Migration of NF-κB
into nucleus
Proteasomal
degradation
RAS MAPKKK
MAPKK
MAPK
Transcription
Migration of MAPK
into nucleus
Keap1
Nrf-2
Nrf-2
Proteasomal
degradation
Nrf-2
Keap1
Dissociation
due to physical
changes
Migration of Nrf-2
into nucleus
Transcription
Inflammatory mediators,
e.g: IL-6, IL-8, COX-2, iNOS
Antioxidants & detoxifying enzymes,
e.g: HO-1, SOD. GPx, NQO1
Inflammatory response Cell defense
Mitochondrion
Mitochondrion
O2O2
O2O2
O2
O2
O2
O2
UV,
radiation
Cigarette smoke
oxidants
Inflammatory
cytokines
Hypoxia
Viruses, bacteria
Ethanol
Activated
receptor
Inducing signal
Carotenoids
Carotenoids
Figure Inflammatory signaling pathways and carotenoids. Under resting conditions, inactive NF-κB is bound to its inhibitory
protein IκB in the cytoplasm. After stimulation with, that is, oxidative stress, inflammatory cytokines, or hypoxia, IκB protein is
phosphorylated by the IKK complex leading to the ubiquitination and proteasomal degradation of the IκB protein. This releases
NF-κB, which can then translocate to the nucleus and the transcription of inflammatory cytokines can start [213]. It could be
hypothesized that carotenoids or their derivatives can interact with cysteine residues of the IKK and/or NF-κB subunits and, as
such, inactivate the NF-κB pathway [93]. Nrf2 is kept inactive by Keap1 in the cytosol by poly-ubiquitination and rapid
degradation through the proteasome. During redox imbalance, the Keap1-Nrf2 association is disrupted and Nrf2 can dissociate
from Keap1, entering the nucleus, and leading to the transcription of antioxidant and detoxifying enzymes, which promotes
cytoprotection [58]. Carotenoids and their derivatives seem to interact with Keap1 by changing its physical properties [214].
Mitogen-activated protein kinases are a family of Ser/Thr protein kinases. MAPK signaling cascades are organized
hierarchically into a 3-kinase architecture. MAPKs are phosphorylated and activated by MAPK-kinases (MAPKKs), which in
turn are phosphorylated and activated by MAPKK-kinases (MAPKKKs). The MAPKKKs are in turn activated by interaction with
the family of small GTPases and/or other protein kinases, connecting the MAPK module to cell surface receptors or external
stimuli [215]. It is not known how carotenoids interact with the MAPK pathway. Part of the images from Motifolio drawing
toolkits (www.motifolio.com) were used, with permission, in the figure preparation.
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signaling pathways and relation to chronic disease preven..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.07.010
protein, κB (eg, IκB-α,IκB-β,IκB-γ,IκB-ε), which resides in the
cytoplasm. Specific (TNF-α,IL-1β), as well as unspecific
(oxidative stress, UV radiation), signals can activate the
NF-κB pathway (Figure), starting with the dissociation of the
inhibitor from the NF-κB-complex and the entrance of NF-κB
into the nucleus. Here, the NF-κB complex can bind to DNA
and activate the transcription of various target genes [56],
many of which are inflammatory and immunoregulatory [57].
Blocking NF-κB activation, anywhere in the cascade, will lead
to a repression of the transcription of the target genes and
thus reduced inflammation.
The Keap1-Nrf2 pathway (Figure) plays an important role
in the cellular defense against endogenous or exogenous
stress caused by ROS [58]. Under normal conditions, Nrf2 is
bound to its repressor protein Keap1 in the cytosol, which
leads to its degradation by ubiquitinylation [59]. Keap1 is a
cysteine-rich protein with 27 cysteine residues, and its
conformation can be modified by different oxidants and
electrophiles, thereby leading to the liberation of Nrf2 and
its translocation to the nucleus [58]. Modifications of the thiol
residues of Keap1 lead either to a disrupted interaction with
Nrf2, which can no longer be ubiquitinlyated, or to a
dissociation of Nrf2 from Keap1. Either way, Keap1 is
inactivated, and the newly synthetized Nrf2 can then translo-
cate to the nucleus. It binds to the antioxidant response
element (ARE), which leads to the expression of antioxidant
and cytoprotective enzymes, for example, heme oxygenase 1
(HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate
cysteine ligase (GCL), or glutathione-S-transferases (GSTs) [59].
Mitogen-activated protein kinases are serine/threonine
kinases. Their functions and regulations have been conserved
from unicellular organisms to multicellular organisms. Mam-
malians have 3 well-characterized MAPK cascades (Figure):
extracellular signal-regulated kinases (ERK1/ERK2), c-Jun N-
terminal kinases (JNK1, JNK2, and JNK3), and p38 kinases [60].
Mitogen-activated protein kinases are typically organized in a
3-kinase architecturethat is activated by various extracel-
lular stimuli, including IL-1β, TNF-α, lipopolysaccharide (LPS),
and oxidative stress [61]. The 3-kinase architecture is com-
posed of MAPK, a MAPK activator (MEK, MKK, MAPK kinase),
and a MEK activator (MEK kinase or MAPK kinase kinase). As
they form a kinase cascade, each downstream kinase serves
as a substrate for the upstream activator [62].
3. In vitro studies
3.1. General considerations and implications of ROS
Many in vitro studies (Table 1), especially those that include
cellular models, have aided in establishing a link between
carotenoids, oxidative stress, and inflammation. There are
several advantages in employing cellular models to investi-
gate the effects of carotenoids on inflammation-related
pathways. These include the ability to investigate, under
well-defined conditions, specified carotenoid concentrations
and specific types of cells and therefore allowing for a large
number of investigations that are suitable for hypothesis
building and studying mechanistic effects.
Most studies have investigated the more abundant carot-
enoids (eg, β-carotene, lutein, and lycopene), and occasionally,
less frequently ingested ones, such as astaxanthin, are
examined. The limitations of these cellular studies include
the difficulty to conduct long-term studies due to limited cell-
life, the missing interactions with other cells present in vivo,
and the way of administration, that is, using carotenoids in
solvents (eg, tetrahydrofuran and dimethylsulfoxide) rather
than in lipoproteins or mixed micelles. This may also have
percussions on their stability and cellular uptake, which
normally occurs either passively or via transporters (SRBI,
CD36, NPC1L1). In addition, many studies have used non-
physiological high concentrations, that is, greater than ca. 2
μM[25] (plasma and tissues) and ca. 20 μM (gut) [63]. Another
controversy exists regarding the way in which inflammation
is stimulated. Tumor necrosis factor α[64], LPSs [6567], IL-1β
[68,69],H
2
O
2
[70,71], and bacterial [72,73] or viral stimulation
[74,75] are among the most common stimuli in vivo. Typically,
several of these factors are involved, activating multiple
rather than individual pathways of inflammation [76].
Several cellular models for studying potential anti-
inflammatory effects of carotenoids have been estab-
lish ed. As carotenoid concentration is highest in the gut, several
studies have focused on gastrointestinal epithelial cells such as
Caco-2 [77], HT-29 cells [78], and human gastric epithelial
adenocarcinoma (AGS) [79]. Because of their implication in
many inflammatory mechanisms, monocytes/macrophages
are another interesting target of carotenoids [80]. Carotenoids
are mainly stored in adipose tissue and adipocytes (3T3-L1)
[81,82], and keratinocytes (human primary keratinocyte [HPK])
[83] have been used in several studies. Retinal pigment
epithelial cells (ARPE-19) may also accumulate in the skin and
in the retina.
Reactive oxygen species could cause inflammation, and
several in vitro studies have shown that β-carotene, lycopene,
and lutein were able to reduce ROS production [84]. Lycopene
has been suggested as a potent compound to decrease ROS,
such as that generated by smoke, and to modulate redox-
sensitive cell targets which include protein tyrosine phospha-
tases, protein kinases, MAPKs, and transcription factors [85].
For instance, lycopene at 2 μM was able to reduce the effect of
smoke on molecular pathways involved in inflammation, cell
proliferation, and apoptosis as well as on carcinogen-activat-
ing enzymes, and it also inhibited the formation of smoke-
induced DNA adducts and smoke-stimulated insulin-like
growth factor signaling [85].Inanotherstudy,2μMof
lycopene significantly reduced ROS levels by 60% in human
monocytes (THP-1) when stimulated with cigarette smoke
[86].Lutein(20μM) and β-carotene (20 μM) showed a
significant reduction (20% and 10%, respectively) of ROS in
AGS cells after stimulation with H
2
O
2
[87]. Similarly, β-carotene
(10 and 20 μM) significantly reduced ROS in AGS cells following
Helicobacter pylori stimulation [88].
Reactive oxygen species can also be produced by poor
coupling of the P450 catalytic cycle [89]. Several carotenoids,
especially lycopene, have been shown to modulate liver
metabolizing enzymes, such as cytochrome P4502E1, thus
resulting in anticancerous activity. In one example, lycopene
reduced ROS production through the interaction with NAD(P)
H oxidase and NOX-4 (a homologue of NAD(P)H) [90].
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signaling pathways and relation to chronic disease preven..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.07.010
Table 1 Effects of carotenoids on different inflammatory mediators investigated in cell cultures
Carotenoids
investigated
Cell type Inflammatory
stimulus
Carotenoid
concentration
Inflammatory effects
a
Reference
Astaxanthin HPKs UVB irradiation
(80 mJ/cm
2
)
8μM NF-κB associated: reduced gene
expression of COX-2 and IL-8,
decreased PGE-2 and IL-8 secretion.
No effect on NF-κB translocation
to the nucleus
Terazawa
et al [83]
MAPK associated: decrease of p38 and
ERK phosphorylation, no effect on JNK
phosphorylation
Astaxanthin U397
(human macrophage-
like cells)
H
2
O
2
(100 μM) 10 μM NF-κB associated: decrease of IL-1β, Il-6
secretion. Blockage of NF-κB nuclear
translocation
Speranza
et al [94]
Astaxanthin ARPE-19
(retinal pigment
epithelia cells)
H
2
O
2
(200 μM) 5-20 μM ROS: reduced level of ROS Li et al [105]
Nrf2 associated: Increased mRNA
expression of NQO1, HO-1, GCLM, and
GCLC. Increased nuclear localization
of Nrf2
Astaxanthin PC-12
(pheochromocytoma
cell line)
MPP (N-methyl-4-
phenylpyridinium;
500 μM)
10 μM ROS: reduced level of ROS by 22% Ye et al [107]
Nrf2 associated: decreased mRNA
expression of NOX-2 by 24%. Increased
mRNA expression of HO-1 by 117%.
Increased Nrf2 protein by 100%
β-Carotene AGS
(human gastric
epithelial cells)
H. pylori
(bacterium/cell
ratio 300:1)
10 and 20 μM ROS: reduced level of ROS Jang et al [88]
NF-κB associated: 90% decrease of
iNOS and COX-2 mRNA expression
and NO and PGE2 protein levels.
Dose-dependent decreased DNA
binding activity of NF-κB, via
prevention of IκB degradation
MAPK associated: inhibition
of p38, ERK, and JNK
phosphorylation
3-hydroxy-β-
damascone
HT-29
(human colon
adenocarcinoma cells)
n.i. 20 and 50 μM Nrf2 associated: induction of NQO1.
Increased Nrf2 transcriptional activity
Gerhäuser
et al [100]
RA SH-SY5Y
(human neuroblastoma
cell line)
n.i. 10 μM Nrf2 associated: Increased mRNA
expression of Nrf2 and NQO1
Zhao et al [101]
ATRA SK-N-SH
(human neuroblastoma
cell line)
n.i 10 μM NF-κB associated: Degraded IκB-βand
increased NF-κB DNA binding activity
Kiningham
et al [206]
RA MCF-7
(human mammary
cancer cells)
n.i. 1 μM Nrf2 associated: decreased binding
activity of Nrf2 to ARE
Wang
et al [103]
β-Carotene
or lutein
AGS
(human gastric
epithelial cells)
H
2
O
2
(100 μM) 20 μM ROS: 10% reduction of ROS by β-carotene
and 20 % reduction by lutein
Kim et al [87]
NF-κB associated: reduced IL-8 mRNA
expression and protein levels. Lutein
10% higher effect. Decreased DNA-
binding activity of NF-κB
β-Carotene or
astaxanthin or
capsanthin or
bixin
K562
(human myelogenous
leukemia cells)
n.i. 5-50 μM Nrf2 associated: up-regulation of
Nrf2 expression
Zhang
et al [98]
Lutein or
zeaxanthin
ARPE-19
(human retinal
pigment epithelial
cells)
A2E
(lipofuscin
fluorophore)
(10 μM)
10 μM NF-κB associated: decrease of IL-8
mRNA expression and protein levels
Bian et al [207]
Lycopene RAW264.7
(murine macrophages)
LPS (1 ng/mL) 0.5; 1; 2 μM NF-κB associated: 30%-40% reduction
of IL-6 and IL-1βmRNA expression
Marcotorchino
et al [82]
MAPK associated: decrease of JNK
phosphorylation (no effect on p38
and ERK1/2 phosphorylation)
(continued on next page)
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3.2. Inflammation factors associated with NF-κB
Several studies have shown that carotenoids can reduce
NF-κB activation [52,86,91,92], although it could be hypothe-
sized that not (only) the intact carotenoids but also their
derivatives are the key players in NF-κB regulation. Carotenoid
derivatives, such as apocarotenals, contain electrophilic
groups that can interact with the cysteine residues of IκB
kinase (IKK) and NF-κB subunits (p65), thereby inactivating the
NF-κB pathway [93].
Whichever the causal agent, it has been shown that
β-carotene at 2 to 20 μM reduced the expression of several
downstream targets of NF-κB, including iNOS and COX-2, by
90% in bacterially infected AGS cells [88]. In the same study,
β-carotene also decreased the secretion of nitric oxide (NO)
and PGE2. Lycopene, at much lower concentrations (0.5-2 μM),
showed similar effects on inflammatory cytokines and
signaling pathways, by reducing IL-6 and Il-1βsecretion by
30% to 40% in LPS-stimulated RAW264.7 cells [82]. Lycopene (2
μM) decreased the IL-6 messenger RNA (mRNA) expression in
preadipocytes (3T3-L1) by 40% and in mature adipocytes
(3T3-L1) by 37% [81], suggesting that it may play an important
role in the homeostasis of adipose tissue. At 2 μM, lycopene
reduced IL-8 mRNA expression and protein levels, as well as
NF-κB DNA binding and the production of ROS in cigarette
smokestimulated human monocytic (and macrophage-like)
cells (THP-1) [86], therefore supporting that the idea that
lycopene may be an interesting candidate for further testing in
vivo, such as in smokers. Another research group analyzed the
effect of astaxanthin on several inflammatory mediators and
NF-κBinH
2
O
2
-stimulated human macrophage-like cells
(U397) and found that it lowered IL-1βand IL-6 secretion and
blocked NF-κB translocation to the nucleus [94]. In addition to
blocking translocation, studies demonstrated that β-carotene
could inhibit H
2
O
2
and H. pyloriinduced NF-κBDNA binding
in the nucleus of AGS [87,88] and macrophage cells [95] at
concentrations of around 2 to 25 μM. This was accomplished
via the prevention of the degradation of the inhibitor IκB[88]
in the cytosol, thus suggesting further downstream general
anti-inflammatory effects.
Some studies have investigated the relationship between
carotenoids and UV-induced inflammation, because caroten-
oids may be distributed in the human skin [96,97]. Studies in
UV-Birradiated HPKs revealed that astaxanthin at 8 μM
reduced COX-2 and IL-8 gene expression and also PGE2 and
IL-8 protein secretion [83].Itwasalsotestedwhether
astaxanthin influenced NF-κB translocation, but no effect
was seen, perhaps due to transient effects of the latter.
Table 1 (continued)
Carotenoids
investigated
Cell type Inflammatory
stimulus
Carotenoid
concentration
Inflammatory effects
a
Reference
Lycopene THP-1
(human monocytic
cell line)
Cigarette
smoke (0.5%)
2μM ROS: reduced ROS production Simone
et al [86]NF-κB associated: decrease of IL-8
mRNA expression and protein levels.
Decrease of NF-κB DNA binding activity
Lycopene RAW264.7
(murine macrophages)
LPS (1 μg/mL) 1-10 μM NF-κB associated: decreased mRNA of
NO and IL-6. Inhibition of NO and IL-6.
Inhibition of IκB and phosphorylation
and degradation and NF-κB translocation
Feng et al [111]
MAPK associated: decrease of p38 and
ERK phosphorylation but no effect on
JNK phosphorylation
Lycopene THP-1
(human monocytic
cell line)
7-ketocholesterol
(25 μM)
2μM ROS: reduced ROS production through
reduction of NOX-4 and NAD(P)H
oxidase.
Palozza
et al [110]
MAPK associated: decrease of p38,
ERK and JNK phosphorylation
Lycopene 3T3-L1
(murine preadipocytes)
TNF-α(15 ng/mL) 2 μM NF-κB associated: decreased mRNA
of IL-6 by 40 %. Inhibition of IKKα/β
phosphorylation
Gouranton
et al [81]
Fucoxanthin BNL CL.2
(mouse hepatic cells)
n.i. 0.5-5 μM ROS: significant increase of ROS Liu et al [104]
Nrf2 associated: increase of nuclear
Nrf2 protein, increased binding of
Nrf2 to ARE. Increased mRNA of
HO-1, NQO1
MAPK associated: increased
phosphorylation of ERK/p38
Lycopene or
β-carotene or
phytoene or
astaxanthin
HepG2
(human hepatocellular
carcinoma cells)
and MCF-7
n.i. 2-50 μM Nrf2 associated: increase of Nrf2-
translocation to the nucleus in
HepG2 cells. Activation of ARE by 3
to 4 fold in MCF-7 and HepG2 cells
(lycopene concentration of 6 μM)
Ben-Dor
et al [51]
Abbreviations: n.i., no information; NO, nitric oxide; GCLM, glutamate cysteine ligase modulatory subunit; GCLC, glutamate cysteine ligase
catalytic subunit; NOX-2/4, NAD(P)H oxidase; NADPH, nicotinamide adenine dinucleotide phosphate.
a
All effects were significant.
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3.3. Inflammation factors associated with Nrf2 and MAPK
Recently, due to the typical strong antioxidant effects of
lycopene, the effects of lycopene on Nrf2 have been reviewed
[90], and it was noted that lycopene was able to up-regulate
the Nrf2 binding ARE system in several cell types such as
MCF-7 breast cancer and HepG7 liver cells [51]. In a study by
Zhang et al [98],β-carotene, astaxanthin, capsanthin, and
bixin at 5 to 50 μM up-regulated the expression of Nrf2 in K562
leukemia cells. In a study by Ben-Dor et al [51] that focused on
MCF-7 cells, expressions of reporter genes fused with ARE
sequences after various carotenoid treatments (lycopene,
phytoene, β-carotene, and astaxanthin) at 2 to 10 μM were
compared. Their results showed that lycopene had the
strongest effect and, hence, suggested that it is a good
activator for the nuclear translocation of Nrf2. This may
seem somewhat surprising due to its low cytosolic solubility
and apolarity, which constitutes a poor activator of Keap1, and
it may shed further doubts on whether lycopene, rather than
its apocarotenal metabolites, are the bioactive constituents.
Because carotenoids such as lycopene lack an electrophilic
group, which appears to be required to react with Keap1, it is
suggested that not the parental carotenoids, but metabolites
following BCO1 and BCDO2 cleavage are the responsible
bioactives [99]. An example includes the 10,10-apocarotenal
derivative of lycopene, which is shown to be active in MCF-7
and prostate adenocarcinoma (LNCaP) cells. In general,
apocarotenals showed higher activity than apocarotenoid
acids in terms of EpRE/ARE transactivation. In another
study, 3-hydroxy-β-damascone, a carotenoid derived polar
flavor (apocarotenoid), induced NQO1 in HT-29 cells after 10-,
20-, or 50-μM exposure [100], with the latter 2 concentrations
being significant. It also appears that retinoic acid (RA), after
cleavage of β-carotene by BCO1, is effective in enhancing Nrf2
translocation. Similarly, in a study on neuroblastoma cells
(SH-SY5Y), RA at 5 to 40 μM induced mRNA expression of Nrf2
and NQO1, dose dependently [101]. Thus, all these studies are
supportive of the idea that more polar carotenoid metabolites
can activate Nrf2 translocation.
It also appears that oxidative stress, caused by rather high
concentrations (>10 μM) of RA, stimulates Nrf2 translocation
[102], whereas lower concentrations (0.1-1.0 μM) may inhibit
Nrf2 translocation, indicating a biphasic activity behavior
[102,103]. However, this is not confirmed in all studies. For
example, in the study by Ben-Dor et al [51], ARE activation did
not depend on the level of intracellular ROS or reduced
glutathione, thus suggesting that this activation was not
related to antioxidant activity. At comparable low concentra-
tions, fucoxanthin (above 0.5 μM) in hepatic cells (BNL CL.2)
increased nuclear Nrf2 protein accumulation, and levels of
mRNA and protein expression of downstream HO-1 and NQO1
[104]. Of note, fucoxanthin also increased ROS levels and
phosphorylated ERK and p38 intracellularly indicating that
prooxidant properties could result in beneficial effects.
In a recent study by Li et al [105], astaxanthin protected
retinal epithelial cells (ARPE-19) from H
2
O
2
induced oxidative
stress by inducing nuclear localization of Nrf2, reducing
intracellular ROS, and up-regulating NQO1, HO-1, glutamate
cysteine ligase modulatory subunit, and glutamate cysteine
ligase catalytic subunit mRNA expression, although astax-
anthin concentrations were comparatively high at 5 to 20 μM.
In the same study, the involvement of P-Act and its
downstream target Nrf2 was further suggested by inhibitors
of P-Act, diminishing the positive effects of astaxanthin in
ARPE-19 cells. The influence on retinal cells has been met with
increasing interest due to the relation of age-related macular
degeneration (AMD) and retinal carotenoid concentrations
[106]. As carotenoids are also able to cross the blood-brain
barrier and are found in brain cells, several cellular models of
neuroprotection exist, such as for Parkinson's disease. In a
recent study on neuronal PC-12 cells [107],astaxanthin
protected against N-methyl-4-phenylpyridiniuminduced ox-
idative stress at 10 μM, reduced intracellular ROS and NOX2,
and increased Nrf2 and HO-1 protein expression; however,
realistic concentrations in this tissue are expected to be lower
(up to 0.5 μM) [108].
Only a few studies have investigated the effect of
carotenoid cellular exposure on targets of the MAPK pathway,
although interactions have been shown for lutein, zeaxanthin,
and lycopene at the MEK activator level in the 3 different
MAPK cascades [109]. It has been demonstrated that astax-
anthin (4 or 8 μM) decreased the phosphorylation of p38 and
ERK, but no effect was found on JNK in human keratinocytes
[83]. Similar effects were seen for β-carotene, preventing the
phosphorylation of ERK, JNK, and p38 [88] in the cytosol. In a
study by Palozza et al [110], lycopene suppressed MAPK
phosphorylation in oxysterol stimulated macrophages and
prostate cancer cells. In another study, lycopene decreased
JNK phosphorylation but showed no effect on p38 and ERK
phosphorylation in LPS-stimulated RAW264.7 cells [82]. This
finding contrasts with an investigation where lycopene
blocked p38 and ERK phosphorylation but did not affect JNK
phosphorylation in RAW264.7 cells [111]. A possible explana-
tion might be the slightly different concentrations of lyco-
pene, which ranged from 0.5 to 2 μM in one [82] and 1 to 10 μM
in the other study [111]. Also, the time of exposure (often
varying between 6 and 48 hours) and the cell passage number
could have affected the results.
3.4. Summary and outlook
Despite the limitations of cellular studies to model complex in
vivo environments, in conjunction with the often high
concentrations of carotenoids that are investigated, several
carotenoids, at concentrations that are physiologically plau-
sible, have indicated that they are able to reduce ROS. Because
of the relation of ROS and inflammation, it is not too
surprising that carotenoids were also found to positively
modulate markers of inflammation and oxidative stress,
especially those related to the NF-κBorNrf2pathway,
respectively. This occurred by blocking NF-κB translocation
to the nucleus and removal of the Nrf2 repressor Keap1. The
strength of the effect across various carotenoids appears
difficult to predict, as studies often differ in many parameters,
which include cell type, incubation time, and concentration,
and the effects may not strictly be related to intracellular
oxidative stress. An important factor deserving further
investigation is the potential carotenoid metabolites which,
also depending on the cell type and the ability to cleave
carotenoids via eg, BCO1 or BCDO2 into apocarotenals, appear
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Table 2 Effects of carotenoids on inflammation in animal models
Animals
involved
Carotenoid
investigated
Concentration
of carotenoid
Stimuli Treatment
length
Tissue/cells
sampled
Main effects Reference
Rats Annatto
extract or
β-carotene
0.1 % in the diet n.i. 7 d Neutrophils ROS: reduced ROS
production. Increased
mRNA expression of
SOD CAT, p22, p47
Rossoni Junior
et al [114]
Rats Lycopene 10 mg/kg bw Sodium fluoride 5 wk Red blood
cells, heart
and brain
ROS: reduced MDA,
total nitrate/nitrite
and glutathione
Mansour
et al [117]
Rats Lycopene 40 mg/kg bw Mercury chloride Single doses Renal tissues ROS: decreased ROS
levels by 16%.
Decreased MDA and
GSH. Increased GPx
and SOD
Yang et al [118]
Rats Astaxanthin 20 mg/kg bw Alloxan 30 d Neutrophils ROS: reduced TBARS
and H
2
O
2
levels
Marin et al [119]
Rats Lycopene 1-4 mg/kg bw Streptozotocin 10 wk Serum NF-κB associated:
reduced TNF-α
production
Kuhad et al [130]
Rats Lycopene 10, 15, and 20
mg/kg bw
n.i. 12 wk Serum NF-κB associated:
reduced VCAM-1,
MCP-1, IL-8
Liu et al [131]
Rats Lutein 1-100 mg/kg bw Streptozotocin Single
injection
Aqueous
humor
NF-κB associated:
inhibited NF-κB
activation. Decrease
of NO, PGE2, IL-6,
TNF-α, CCL2 and
CXCL2
Kijlstra et al [134]
Rats Fucoxanthin 0.1, 1 or 10 mg/
kg bw
LPS Single
injection
Aqueous
humor
NF-κB associated:
reduced PGE2, NO,
TNF-αlevels
Shiratori
et al [136]
Rats Lycopene 1.1 or 3.3 mg/kg
bw
Alcohol 11 wk Plasma
and liver
NF-κB associated:
induction of CYP2E1
protein and TNF-α
mRNA
Veeramachaneni
et al [143]
Rats Lycopene 6 mg/kg bw Cisplatin Single
injection
Kidney cells ROS: increased levels
of SOD, GPx and CAT.
Sahin et al [145]
NF-κB associated:
decrease of NF-κB
expression.
Nrf2 associated:
increased Nrf2 and
HO-1 expression
Rats Astaxanthin 25 mg/kg bw Cyclophosphamide 10 d Liver Nrf2 associated:
increase of NQO-1
and HO-1.
Tripathi
et al [146]
Rats Crocin 10 mg/kg bw Cyclophosphamide 6 d Liver and
serum
ROS: increase in
SOD, CAT, GSTs, GPx.
Jnaneshwari
et al [138]
Rats Crocin 10 or
20 mg/kg bw
Freund's complete
adjuvant
14 d Serum,
liver, and
spleen tissue
NF-κB associated:
decreased IL-1β, IL-6,
TNF-αand COX-2
and PGE2 levels
Hemshekhar
et al [139]
ROS: ROS decrease
by 98 %. Reverted
GSH levels
Rats Crocetin 50 mg/kg bw hemorrhagic shock Single
administration
Renal blood NF-κB associated:
decreased NO,
TNF-αand IL-6
Wang et al [140]
Rats β-Carotene 0.05 % in diet azoxymethane 33 wk Colonic
mucosa
NF-κB associated:
slight reduction of
COX-2 expression
Choi et al [141]
Mice β-Carotene
or lycopene
0.5 g/kg bw Cigarette smoke 4 wk Lung ROS: regulated
expression of
cytochrome P450
Aung et al [126]
Mice β-Carotene 0.6 % in diet n.i. 10, 15,
or 20 wk
Liver NF-κB associated:
reduced mRNA of
Harari et al [127]
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Table 2 (continued)
Animals
involved
Carotenoid
investigated
Concentration
of carotenoid
Stimuli Treatment
length
Tissue/cells
sampled
Main effects Reference
VCAM-1, Il-1α,
MCP-1, IFN-γ
Mice β-Carotene 150 mg/kg bw n.i. 14 wk Lung, liver,
and white
adipose tissue
NF-κB associated:
decreased expression
of genes involved in
interferon production/
regulation
Van Helden
et al [128]
Mice RA 0.5 mg/kg bw n.i. 9 wk CD4(+) cells
and arthritic
joints
NF-κB associated:
decreased IL-17,
IL-6, IL-1βand iNOS
expression. Down-
regulated NF-κB
Kwok et al [92]
Mice Lycogen 1 mg/kg bw Dextran
sodium
sulfate
6 d Plasma NF-κB associated:
reduced expression
of TNF-αand IL-1β
Liu et al [129]
Mice Astaxanthin 1, 10 or
100 mg/kg bw
n.i. 3 d, injections Retinal
pigment
epithelium-
choroid tissue
NF-κB associated:
reduced IL-6, VEGF,
MCP-1 and ICAM-1.
Suppression of NF-κB
activation,
translocation
and IκB degradation
Izumi-Nagai et al
[137]
Mice Lutein 1, 10, and
100 mg/kg
LPS Single
administration
Aqueous
humor and
iris-ciliary
body
NF-κB associated:
dose dependent
reduction of
NO, TNF-α, IL-6,
PGE2 concentration;
NF-κB activation
Jin et al [135]
Mice Lutein 0.1 % in diet Streptozotocin 1 mo Retina ROS: reduced ROS
production
Sasaki et al [120]
MAPK associated:
inhibit ERK activation
Mice Astaxanthin 100 mg/kg bw H
2
O
2
4 times Retina ROS: reduced retinal
damage and oxidative
DNA damage
Nakajima et al
[121]
Mice Vitamin A
and RA
10:1 molar
ratio
Hyperoxia 7 or 14 d Lung Nrf2 associated:
decreased DNA
damage. Reduction
of Nrf2 proteins.
Decrease of
INF-γand macrophage
inflammatory protein
2-αmRNA
James et al [147]
Mice RA 0.5 mg/mouse n.i. n.i. T cells Other pathways:
suppression of IFN-γ
producing CD4(+) and
CD8(+) T cells.
Suppression of STAT4
expression
Van et al [152]
Mice RA 400 μg/mouse Ovalbumin Single
administration
Lymphocytes Other pathways:
inhibition of Th-2 cells
and Th17 related
cytokines
Wu et al [208]
Mice Lycopene 8 or
16 mg/kg bw
Ovalbumin 3 d Lung Other pathways:
decreased IL-4 mRNA.
Increased IFN-γand
T-bet mRNA
Lee et al [156]
Mice Vitamin A 250 IU/g diet Ovalbumin 3 wk Lung Other pathways:
decreased Il-4 and
IL-5 release
Schuster et al
[209]
Mice Lycopene 100 mg/kg bw n.i. Single dose Different
organs
Other pathways:
induction of RARE-
mediated cell signaling
Aydemir et al
[153]
(continued on next page)
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to be in part responsible for the observed anti-inflammatory
reactions. This is plausible given that free carotenoid concen-
tration in the cytosol (where Nrf2 and NF-κB reside) are
typically low and require binding to cysteine residues of these
transcription factors, therefore favoring polar compounds.
Also, carotenoid concentration in such studies is likely to
influence the results. Although Nrf2 translocation appears to
be more prominent at rather higher concentrations (>1 μM),
possibly via increasing oxidative stress due to prooxidant
effects of carotenoids or their metabolites, lower concentra-
tions may have rather inhibitive effects on Nrf2.
4. Animal studies
4.1. General aspects and implications of ROS
Compared with cellular trials, animal models (Table 2) allow
for studying the effects under more complex, that is, realistic,
physiological conditions. Compared with human studies, they
are easier to coordinate under more standardized conditions
and allow for a higher accessibility to tissues. As a drawback,
many animals metabolize carotenoids differently from
humans [112]; their cleavage rate by BCO1 and BCDO2, such
as in mice (being much higher), can be quite altered compared
with humans. In addition, in many animal models, caroten-
oids were administered at supraphysiological doses that were
comparable to ca. 6 mg/kg body weight (bw) when setting 0.5
mg/kg bw in humans as a limit for a physiological dose,
applying the human equivalent dose formula for comparing
doses across species [113]. Animal models resembling the
human carotenoid metabolism include Mongolian gerbils and
various types of monkeys [112].
By providing foods rich in carotenoids, several studies have
aimed to reduce ROS and their negative impact on inflamma-
tion. For example, annatto extract or β-carotene added at 0.1%
to the diet of rats for 7 days decreased ROS production in
neutrophils and increased mRNA concentrations of superox-
ide dismutase (SOD), catalase (CAT), p22(phox), and p47(phox),
which are components of the electron transfer elements of
nicotinamide adenine dinucleotide phosphate oxidase [114].
However, most studies investigated isolated carotenoids
that were added to the diet. Administration of lycopene in
isolated form has received some attention because of its
strong antioxidant effects in vitro [115]. Similar to cellular
studies, lycopene ameliorated the negative effects of ROS
produced during anticancer chemotherapy [116]. In another
study on rats, daily lycopene administration of 10 mg/kg bw
for 5 weeks normalized oxidative stress (caused by sodium
fluoride exposure), assessed as plasma MDA and total nitrate/
nitrite [117]. Lycopene also ameliorated oxidative stress in
tissues, for example, in renal tissues (glutathione [GSH], MDA,
SOD), when administered as a single dose (40 mg/kg bw) prior
to mercury poisoning [118].
Astaxanthin (20 mg/kg), administered to diabetic rats for 30
days, reduced oxidative stress (measured by thiobarbituric
acid reactive substance, H
2
O
2
) in neutrophils, although it did
not go back to basal levels [119]. In another diabetes model,
lutein (0.1% in the diet) in rats prevented ROS-related retina
degeneration after 4 months of supplementation [120].
Positive effects on the retina were also found for astaxanthin
in mice, but they received very high doses, that is 4 times 100
mg/kg [121]. These results support the positive effects of
carotenoids for eye health. In larger animals, for example,
dogs, 20 mg/d of astaxanthin for 16 weeks improved mito-
chondrial function and markers of oxidative stress, including
plasma glutathione peroxidase (GPx) and NO [122]. These
observations were attributed, in addition to indirect effects on
intracellular signaling (especially Nrf2) to the following: (a)
quenching of singlet oxygen, (b) reaction with free radicals, (c)
acting as an antioxidant via reactivating vitamin E or vitamin
C, or (d) reducing DNA damage [123]. Further additional modes
of action may exist. Several animal studies have suggested
that carotenoids can up-regulate the P450 cytochrome mono-
oxygenase family and may come into play when detoxifying
Table 2 (continued)
Animals
involved
Carotenoid
investigated
Concentration
of carotenoid
Stimuli Treatment
length
Tissue/cells
sampled
Main effects Reference
Mice Lycopene 0-20 μM LPS Single
administration
Endothelial
cells
Other pathways:
inhibition of HMGB1
release and HMGB1-
mediated TNF-secretory
phospholipase A2-IIA
and HMGB1-mediated
proinflammatory
responses
Lee et al [210]
Dogs Astaxanthin 20 mg/d n.i. 16 wk Leukocytes ROS: improved
mitochondrial function
and plasma GPx and
NO levels
Park et al [122]
Chicken Astaxanthin 100 ppm LPS 2 wk Liver and
spleen
NF-κB associated:
increase in iNOS,
IL-1, IL-6 and IFN-γ
mRNA expression
Takahashi
et al [142]
Abbreviations: n.i., not investigated; CCL2, chemokine (C-C motif) ligand 2; CXCL2, chemokine (C-X-C motif) ligand 2; IFN-γ, interferon γ; VEGF,
vascular endothelial growth factor; RARE, RA response element; TBARS, thiobarbituric acid reactive substance.
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xenobiotic compounds. For instance, several enzymes such as
P450E1 in rat liver [124] or CYP 1A1/2 2B1/2 and 3A [125] were
up-regulated in a dose-dependent manner by lycopene, and
both lycopene and β-carotene activated the P450 A1 gene in
mice [126].
4.2. Implication of the NF-κB pathway
In cellular studies, NF-κB or downstream cytokines have been
among the most studied markers of inflammation in animal
studies after carotenoid exposure. Positive effects on inflam-
matory genes, such as those implicated in the expression of
IL-1α, INF-γ, monocyte chemotactic protein 1 (MCP-1), and
vascular cell adhesion protein 1 (VCAM-1), were found after
feeding a diet of 0.6% β-carotene (50% in 9-cis form) to rats (in
which atherosclerotic lesions were induced by a high-fat diet)
for 11 weeks [127]. In another study with BCO1-deficient mice,
14-day β-carotene supplementation (150 mg/kg in diet)
reduced mRNA expression of proinflammatory genes in-
volved in interferon production/regulation in the lung, liver,
and white adipose tissue [128]. This suggests that effects were
independent from vitamin A active compounds, although
BCDO2 may still have been active. As with cellular models,
metabolites of carotenoids have been shown to be effective in
altering the inflammatory processes. In arthritis mice models
[92], RA intraperitoneal (IP) at 0.5 mg/kg for 9 weeks reduced
several inflammatory parameters, including IL-17 in CD4(+) T
cells and serum IgG/IgG2, down-regulated NF-κB in CD4(+) T
cells, and reduced iNOS, IL-6, and IL-1βin tissues.
Carotenoids other than β-carotene also received some
attention. In a rodent study, colitis induced in mice by
dextransodium sulfate was reduced by the administration
of lycogen (at 1 mg/kg for 6 days), which is a trademark extract
rich in carotenoids. Both TNF-αand IL-1βin plasma were also
reduced [129]. In diabetic induced rats, lycopene given at 1 to 4
mg/kg for 10 weeks reduced inflammation, as measured by
TNF-αin serum, and limited cognitive decline, as indicated by
the Morris water maze test [130]. In a hyperhomocysteinemic
rat model, lycopene at 10, 15, and 20 mg/kg taken for 12 weeks
reduced serum markers of inflammation such as VCAM-1,
MCP-1, and IL-8, thus indicating antiatherogenic effects [131].
Because AMD is related to inflammation processes [132],and
lutein and zeaxanthin have been especially related to AMD [133],
many animal models focusing on the retina have investigated
the potential benefits of carotenoids. As reviewed by Kijlstra
[134], lutein intravenous injections (doses between 1 and 100 mg/
kg) reduced aqueous humor NO, PGE2, IL-6, TNF-α,various
chemokines, and NF-κB activation in the Iris ciliary body in a rat
model, when administered prior to endotoxin induced uveitis
[135]. In a similar rat model of LPS-induced uveitis, fucoxanthin
injected intravenously at 0.1, 1.0, or 10 mg/kg after LPS
administration reduced PGE2, NO, and TNF-αconcentration in
the aqueous humor [136]. In a mouse model of AMD, astaxanthin
given ip to mice (10 or 100 mg/kg bw) reduced NF-κBactivation
and inflammation-related molecules such as vascular endothe-
lial growth factor, IL-6, ICAM-1, and MCP-1 in retinal pigment
epithelium tissues [137]. Taken together, these results suggest
that inflammatory processes in the retina involved in AMD may
be altered by considerable doses of carotenoids.
A further hint toward the bioactivity of polar carotenoids
and their metabolites comes from studies with crocin. This
natural, more polar carotenoid attenuated cyclophosphamide
induced hepatotoxicity in rats after a 6-day administration of
10 mg/kg. In addition to various markers of oxidative stress,
inflammatory markers that included cytokines were signifi-
cantly decreased [138]. In a rat model of arthritis (induced by
Mycobacterium tuberculosis), 10 or 20 mg/kg crocin for 14 days
reduced plasma levels of inflammatory markers, including
TNF-α, IL-1β, IL-6, COX-2, and PGE2, as well as markers of ROS,
such as GSH [139]. In rats, crocin administered at 50 mg/kg
after hemorrhagic shockinduced acute renal failure reduced
negative effects by decreasing NO, TNF-α, and IL-6 and also
attenuating NF-κB expression [140].
Some conflicting results exist and deserve to be mentioned
because there is the possibility of severe underreporting of
nonsignificant or even negative results. In a study by Choi et al
[141],β-carotene given at 0.5 % for 33 weeks to rats with
induced colon carcinogenesis failed to reduce inflammatory
markers such as COX-2. In a broiler study by Takahashi et al
[142], the addition of astaxanthin at 100 ppm to the diet
showed no positive effect on inflammation, but it aggravated
LPS-stimulated inflammation, which was measured by mRNA
expression of iNOS, IL-1, IL-6, and IFN-γin the liver. The
reasons for these observations remain unknown, but altered
metabolism of carotenoids in birds, low dosing of carotenoids,
or overlayering effects with the co-consumed corn (also
containing carotenoids) could have played a role.
Even negative effects of carotenoids have been reported.
When feeding physiological doses of 1.1 or 3.3 mg/kg bw
(equivalent to ca. 45 mg/kg in a 70-kg human) for 11 weeks to
rats receiving high doses of ethanol, the higher lycopene dose
increased hepatic TNF-αmRNA, thus suggesting a prudent
approach when administering carotenoids when chronic
alcohol abuse is involved [143]. Although the reasons remain
unknown, in the same study, it was suggested that alcohol
slowed down BCDO2 enzymatic activity and the formation of
lycopene metabolites. The resulting elevated tissue concen-
trations of the native carotenoid could have caused prooxi-
dative effects [144].
4.3. Implication of the Nrf2 and other pathways
As the importance of Nrf2 in oxidative stress and inflamma-
tion related pathways has only recently been discovered, very
few carotenoid studies investigating Nrf2 have been conduct-
ed in animals. As cancer chemotherapy may cause oxidative
stress, a few studies have attempted to ameliorate this
undesired side effect. In a study with cisplatin (used in
chemotherapy), lycopene ameliorated nuclear Nrf2 and HO-1
decreases in rats receiving 6% lycopene and 1.5% other
carotenoids at 6 mg/kg (phytoene, phytofluene) in their diet
for 6 days (ie, 360 μg/kg final lycopene) [145]. Expression of
NF-κB p65 was also reduced, and the formation of down-
stream SOD, GPx, and CAT was up-regulated in kidney cells. In
another rat study, astaxanthin increased NQO-1 and HO-1
expression in rats, after exposure to either cyclophosphamide
with or without pretreatment of 25 mg/kg astaxanthin [146].In
line with cellular studies, vitamin A active carotenoid
metabolites (ie, RA) improved hyperoxia induced depressed
11NUTRITION RESEARCH XX (2014) XXXXXX
Please cite this article as: Kaulmann A, Bohn T, Carotenoids, inflammation, and oxidative stressimplications of cellular
signaling pathways and relation to chronic disease preven..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.07.010
lung function in mice, reduced DNA damage, protein oxida-
tion, interferon-γ, and macrophage inflammatory protein-2α
mRNA, thus reducing Nrf2 proteins [147].
In addition to primary inflamed cells, it should not be
overlooked that cytokines may also attract cells of the
immune system, such as macrophages, which can then
further stimulate inflammation responses. Carotenoids may
also have effects on these attracted immune cells. It is well
acknowledged that RA and other vitamin A active compounds
are required for optimal functioning and maturation of the
immune system [148], and studies have shown that at a later
age, they can have pronounced effects on the immune system,
such as on CD4(+) and CD8(+) T cells, for example, reducing
their inflammatory potential [149,150]. However, it is possible
that pathways other than NF-κB and Nrf2 also play a role, such
as signal transducers and activators of transcription (STAT)
[151]. For example, in a model of type 1 diabetes, all-trans
retinol at 0.5 mg/mouse reduced STAT4 expression required
for IFN-γproduction and immune cell response [152]. These
activities of RA are of interest because other carotenoid
metabolites may also show similar behavior. In a mouse
study by Aydemir et al [153], it was shown that lycopene can
also activate the RA receptor (RAR) and the RA response
element. Similar results were found by Harrison et al [154].
This study demonstrated that various β-apocarotenoids were
able to bind to RARα, RARβ, and RARγreceptors, thereby
opening the door to many immune-related reactions of
carotenoid metabolites.
Possibly due to similar activities, swelling and inflammation
of the paw in mice (induced by carrageenan) receiving a pepper
carotenoid extract (a mixture of carotenoids, ie, β-carotene,
violaxanthin, capsanthin, and capsorubin) were significantly
reduced at 5, 20, or 80 mg/kg [155]. Such reduced involvement of
inflammatory stimulating cells was also seen after lycopene
administration. In a murine model of asthma (induced by
ovalbumin), lycopene (8 or 16 mg injected IP for 3 days) reduced
inflammation, as measured by infiltration of inflammatory
cells (neutrophils, eosinophils, lymphocytes, and macro-
phages), into the peribrochilar and perivascular regions. It
also decreased mRNA levels of IL-4 in bronchoalveolar lavage
fluid but increased IFN-γand T-bet (encoding for transcription
factors responsible for development and also for IFN-γ
production), thus suggesting an overall anti-inflammatory
activity in upper airway diseases [156]. Lycopene administra-
tion (0-20 μM) also reduced permeability and migration
of leukocytes to the peritoneal cavity in mice. This was
explained by its influence on the high-mobility group box 1
(HMGB1), which is a nuclear protein responsible for the
mediation of proinflammatory stimuli such as of multiple
cytokines and chemoattraction of stem cells [157]. Further data
on the effect of carotenoids on this protein are limited though.
4.4. Summary and outlook
A large number of animal trials have strongly suggested the
implication of NF-κB, Nrf2, and additional transcription
factors, such as STAT, in anti-inflammatory effects of
carotenoids, and thereby influencing many downstream
targets such as cytokine formation. Furthermore, the effects
were correlated with results from several studies, several
studies focusing on the decreased activation of proinflamma-
tory cell types such as CD4(+), CD8(+), and neutrophils, and
possibly even involving (via their metabolites) other nuclear
receptors such as RAR. This represents a novel area to be
further scrutinized. Although often supraphysiological doses
were used to achieve this response, there are also indications
that lower and rather chronically applied concentrations are
likewise effective, although more studies are needed in this
domain. An interesting approach in using in vivo models could
be the use and development of novel, more water-soluble
carotenoids, such as crocin and disodium disuccinate astax-
anthin. Disodium disuccinate astaxanthin has been investigat-
ed for cardioprotection and has been shown to reduce some
inflammatory (and oxidative stress) markers (eg, prostaglandin
F2α[PGF2α], 5-hydroxyeicosatetraenoic acid, 8-isoprostane F2α)
in a peritoneal mouse inflammation model, although doses
were extremely high (500 mg/kg for 7 days) [158].
5. Human studies
5.1. Markers of inflammation and oxidative stress and
general aspects
Human intervention studies (Table 3), especially when
conducted as a randomized, double-blind, and placebo-
controlled design, are still considered the gold standardin
nutritional sciences for testing health effects of dietary
compounds. Among other reasons, studies that include
disease outcome add much more evidence toward proving a
potential relationship between carotenoid consumption and
disease incidence. However, with respect to studying second-
ary plant compounds including carotenoids, human studies
are faced with a dilemma. When administering carotenoid-
rich foods, such as certain fruits and vegetables, many
confounding factors prevail that include dietary fiber, min-
erals, and vitamins, which are also present in the food matrix.
However, when giving individual compounds such as in the
form of supplements, synergistic effects between carotenoids
and micronutrients (eg, between vitamin C and vitamin E),
aspects of overdosing, or altered bioavailability (missing food
matrix aiding eg, in emulsification) may result in different
release kinetics, uptake, and biodistribution than carotenoids
bound to the food matrix.
An indirect indication of the potential health benefits of
carotenoids may be found in epidemiologic studies, although
aspects of inflammation in such studies are not typically
assessed. In addition, epidemiologic studies cannot demon-
strate causality. Furthermore, due to a large number of
confounding factors, it is difficult to predict the effect of
individual food constituents. Nevertheless, a number of large-
scale prospective cohort studies such as the EPIC study [159],
the Los Angeles Artheriosclerosis Study [160], and the study by
Kabagambe et al [161] have produced interesting and robust
data with respect to hard end points.Most of these studies
have been reviewed in further detail elsewhere [84,162]. They
suggest that there are significant health benefits when
consuming 3 to 8 servings of fruits and vegetables per day,
whereas a lower number of servings often showed to be
12 NUTRITION RESEARCH XX (2014) XXXXXX
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Table 3 Effects of carotenoids or carotenoid rich food items on inflammatory mediators and oxidative stress in humans
Study type Cell type or
specimen
Test meals Time frame No. of
subjects
Carotenoids
investigated
Aspects
studied
Main effects Reference
Intervention
studies with
food items
PBMCs Fruits and
vegetables
(2 or 8
servings/d)
8 wk 64 men n.i. Fruit/
vegetable
consumption
and
immunologic
markers
Consumption of
fruits/vegetables:
no impact on
TNF-α, IL-12
(2 and 8 servings/d)
and C-reactive
protein
(2 servings/d).
Consumption of
8servings/d
lowered C-reactive
protein
Watzl
et al [198]
Urine and
serum
Vegetables
(2, 5, or 10
servings/d =
130 g, 237 g,
614 g)
3 wk 49 women n.i. Vegetable
consumption
and oxidative
stress,
inflammation
Consumption of
vegetables: no
effect on urinary
8-isoprostane F2α
and serum C-
reactive protein
Crane
et al [199]
PBMCs 68 g avocado 1
consumption
11
individuals
n.i. Avocado and
inflammatory
responses
Avocado
consumption
reduced IL-6
secretion and
NF-κB activation
Li et al
[211]
Fruits and
vegetables
12 y 73286
women
α-and β-
carotene,
lycopene,
lutein/
zeaxanthin β-
cryptoxanthin
Dietary
carotenoids
and CAD
Significant inverse
relation between
intake of β-carotene
and α-carotenerich
foods and coronary
artery disease
Osganian
et al [212]
Carotenoid-
rich foods
(mostly
carrot)
15 y 559 men α-carotene,
β-carotene
Carotenoid-
rich foods
and CVD
α- and β-carotene
rich food: lowered
CVD mortality. Other
carotenoids: no effect
Buijsse
et al [165]
Tomato
products
––Lycopene Tomato and
prostate
cancer
Tomato and tomato
products might play
a role in prostate
cancer, when highly
consumed
Etminan
et al [166]
Food rich in
carotenoids
7-16 y 399 765
individuals
α-and β-
carotene,
lycopene
lutein/
zeaxanthin, β-
cryptoxanthin
Carotenoid-
rich food and
lung cancer
Intake of β-
cryptoxanthinrich
food: inversely
associated with
lung cancer risk
Mannisto
et al [167]
White blood
cells
Tomatoes
(360-728 g/d)
1 single dose 5
individuals
n.i. DNA
oxidation
Tomato consumption
decreased levels of
8-hydroxyguanine
Rehman
et al [180]
Peripheral
lymphocytes
Tomato juice
(330 mL/d
3-4 wk)
8 wk in total 23 men Lycopene,
β-carotene,
α-carotene
lutein
DNA
oxidation
Reduction of strand
breaks in lymphocyte
DNA. Reduction of
oxidative base damage
measured via COMET
assay through the
carrot juice
Pool
et al [178]
Carrot juice
(330 mL/d,
5-6 wk)
Dried spinach
in milk/water
(10 g/d,
7-8 wk)
Urine Tomato
products
3 wk 12 women Lycopene Tomato
products and
markers of
lipid
oxidation
Significant reduction
of 8-iso-PGF2αdue to
tomato products
consumption
Visioli
et al [181]
Leucocytes Tomato 3 wk 32 men Lycopene Tomato Tomato sauce Chen
(continued on next page)
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Table 3 (continued)
Study type Cell type or
specimen
Test meals Time frame No. of
subjects
Carotenoids
investigated
Aspects
studied
Main effects Reference
and prostate
tissue
sauce (30 mg
lycopene/d)
sauce and
prostate
cancer
reduced oxidative
DNA damage in
leucocytes and in
prostate tissue
et al [182]
Plasma Tomato juice
(500 mL/d)
4 wk 57 diabetic
subjects
Lycopene Tomato juice
and LDL
oxidation
Tomato juice
consumption
increased the
resistance of LDL
to oxidation
Upritchard
et al [184]
Skin Tomato paste
(55 g/d
including
16 mg
lycopene)
12 wk 20 women Lycopene Tomato paste
and
cutaneous
photodamage
Protection against
cutaneous
photodamage
through reducing
mitochondrial DNA
damage
Rizwan
et al [186]
PBMCs Tomato juice
(330 mL/d,
47.1 mg
lycopene)
8wk 53
individuals
Lycopene Tomato juice
and cell-
mediated
immunity
Tomato juice
decreased IL-2
and IL-4 secretion
Watzl
et al [191]
Whole blood Tomato drink
(5.7 mg
lycopene, 3.7
mg phytoene,
2.7 mg
phytofluene, 1
mg
β-carotene,
1.7 mg
α-tocopherol)
26 d 26
individuals
Lycopene Tomato-
based drink
and markers
of
inflammation
Tomato-based drink
decreased TNF-α
levels
Riso et al
[192]
Serum Tomato paste
(200 g/d, 16
mg lycopene);
pure lycopene
(16 mg/d)
1 wk 30 men Lycopene Tomato paste
and lycopene
and different
target genes
of prostate
cancer cells
Tomato paste:
up-regulated IGFBP3
and Bax/Bcl-2 ratio
and down-regulated
cyclin D1, p53 and
Nrf2. Lycopene
con-sumption
up-regulated IGFBP3,
c-fos, and μPAR.
Talvas
et al [200]
Lymphocytes Passata sauce
(170 g pasta,
ca. 7 mg/d
lycopene)
3 wk 24 men Lycopene Passata sauce
on oxidative
stress
Consumption of
passata sauce: no
effect on HO-1 in
lymphocytes
Markovitch
et al [201]
Intervention
studies with
isolated
carotenoids/
suppl.
β-Carotene (20
mg/d) and
vitamin E (50
mg/d) suppl.
8 y 29133
smokers
and
nonsmokers
β-Carotene β-Carotene
and vitamin E
and lung
cancer
Increased lung cancer
risk in smokers
Albanes
et al [172]
β-Carotene (30
mg/d) and
retinyl
palmitate
(25000 IU)
suppl.
5 y 18 314
smokers
and
nonsmokers
β-Carotene β-Carotene
and vitamin
A and lung
cancer
incidence
Increased lung cancer
incidence in smokers
Omenn
et al [171]
β-Carotene (20
mg/d) and
vitamin E (50
mg/d) suppl.
6 y 28 519 men β-Carotene Effect on
stroke
incidence in
smokers
Increased risk of
intracerebral
hemorrhage
Leppälä
et al [174]
vitamin C (120
mg/d) and E
(30 mg/d) and
β-carotene (6
mg/d) and Se
(100 μg/d) and
Zn (20 mg/d)
7.5 y 13 017
individuals
β-Carotene Effect on
cancer
incidence
and all-cause
mortality
Decrease in total
cancer incidence and
all-cause mortality in
men but not in women
Hercberg
et al [177]
14 NUTRITION RESEARCH XX (2014) XXXXXX
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signaling pathways and relation to chronic disease preven..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.07.010
ineffective [163,164]. Likewise, several prospective studies
have suggested that carotenoid consumption in the diet was
associated with a reduced risk of cardiovascular mortality
[165], or developing type 2 diabetes [23], prostate cancer [166],
and lung cancer [167]. Similar results were also found for
tissue levels and chronic diseases [168].
Table 3 (continued)
Study type Cell type or
specimen
Test meals Time frame No. of
subjects
Carotenoids
investigated
Aspects
studied
Main effects Reference
suppl.
β-Carotene (15
mg/d) and Se
(50 μg/d) and
vitamin E (30
mg/d)
5.25 y 30 000
individuals
β-Carotene Effect on
cancer
incidence
Decrease in total
mortality and in
cancer mortality
Blot et al
[176]
β-Carotene
(17.8 mg/d)
3.3 y (mean) 232 606
individuals
β-Carotene β-Carotene
and CVD
β-Carotene
supplementation
increased slightly the
risk to develop CVD
Bjelakovic
et al [169]
β-Carotene
(4 μg/d) or
lutein 1.5 μg/d
10 y 77 126
individuals
β-Carotene
lutein
Effect of
carotenoids
on lung
cancer
β-carotene: increased
risk of small cell lung
cancer. Lutein
supplementation:
increased risk of non
smallcelllungcancer
Satia et al
[173]
Lutein
(20 mg/d)
3mo 65
individuals
Lutein Lutein, serum
cytokines,
lipid profile
Supplementation with
lutein: decreased
levels of IL-6 and MCP-
1, also of LDL and TG
Xu et al
[196]
Plasma Lycopene
(6 or
15 mg/d)
8 wk 126 men Lycopene Lycopene and
endothelial
health,
oxidative
stress
Lycopene
supplementation
(15 mg/d): increased
SOD plasma activity,
decreased lymphocyte
DNA comet tail length
Kim et al
[183]
Plasma and
urine
Lycopene
(0,6.5, 15
and 30 mg/d)
8wk 77
individuals
Lycopene Lycopene and
biomarkers of
oxidative
stress
Lycopene
supplementation: no
effect on urinary
F2-isoprostanes, MDA,
LDL oxidation.
30 mg/d: decreased
lymphocyte DNA
damage and urinary
8-OHdG
Devaraj
et al [187]
Serum Lycopene
(10 mg/d)
2 mo 35 diabetic
patients
Lycopene Lycopene and
oxidative
stress
Lycopene
supplementation: no
effect on antioxidant
capacity, oxidized LDL,
but increased MDA
Neyestani
et al [188]
Plasma Lycopene
(12 mg/d)
56 d 37 women Lycopene Lycopene and
lymphocyte
DNA damage
Lycopene
consumption reduced
DNAdamagein
lymphocytes but did
not prevent it.
Zhao et al
[189]
Plasma Lycopene
(15 mg/d)
1wk 8
individuals
Lycopene Lycopene and
lymphocyte
DNA damage
Lycopene
consumption
prevented DNA
damage in
lymphocytes.
Torbergsen
et al [190]
Prostate
cancer
Lycopene
(15 mg/d)
3 wk 26 men Lycopene Lycopene and
prostate
cancer
Lycopene
consumption: no effect
on apoptotic markers
Kucuk et al
[193]
Plasma Lycopene 1 wk 18 men and
9 women
Lycopene Lycopene and
biomarkers of
endothelial
health
Lycopene: no effect on
MDA levels
Denniss
et al [194]
Abbreviations: n.i., not investigated; suppl., supplements; CHD, coronary heart disease; 8-OHdG, 8-oxo-2deoxyguanosine; CAD, coronary artery
disease; TG, triglycerides.
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In contrast to epidemiologic studies that assess the
relationship of whole fruits/vegetables and their positive
effects on chronic diseases, many intervention studies with
isolated carotenoids have failed to demonstrate health bene-
fits. Instead, they appear to show that the risk for developing
CVD [169], lung cancer [170173],orstroke[174] increased.
However, a few trials have revealed beneficial effects after
carotenoid supplementation, especially when given to popula-
tions marginally def icient in carotenoid s [175177], highlighting
that administration of potential prooxidants to nonhealthy
subjects bears several risks.
5.2. Carotenoids, oxidative stress, NF-κB, Nrf2,
and other pathways
Compared with in vitro studies, it is more difficult to measure
the antioxidant effects of carotenoids in vivo, as many factors
govern the antioxidant system [84] and only small or transient
effects are expected (Table 3). Pool et al [178] showed that
consuming 330 mL tomato juice (40 mg lycopene) or 330 mL
carrot juice (containing 22 mg β-carotene and 16 mg α-carotene)
or consuming 10 g dried spinach powder in water/milk resulted
in a significant decrease in endogenous levels of strand breaks
in lymphocyte DNA. The carrot juice intervention was the only
intervention in this study that significantly reduced oxidative
damage. In another carrotjuice intervention[179], 240 mL carrot
juice for 3 weeks provided to breast cancer survivors signifi-
cantly reduced 8-iso PGF2α, although the study was
without a control group. Another study by Rehman et
al [180] (5 individuals) showedthat a single serving of tomatoes
(360-728 g/d) reduced oxidative DNA base damage level in white
blood cells within 24 hours. It has also been shown that the
intake of tomato products for 21 days decreased urinary
8-iso-PGF2αby 50% [181]. The consumption of lycopene-rich
tomato products (sauce, paste, or juice) for 4 to 8 weeks
decreased oxidative DNA [182] and lymphocyte DNA damage
in healthy patients [183], and it increased the resistance of
low-density lipoprotein (LDL) to oxidation in diabetic patients
[184], similar to another study [185]. Lycopene-rich tomato paste
also protected against cutaneous photodamage by reducing
mitochondrial DNA damage [186].
Some positive, but more mixed results were seen in studies
including carotenoid supplements. Although taking lycopene
(6.5, 15, 30 mg/d) in pure form for 8 weeks had no effect on
urinary F2-isoprostanes, MDA, and LDL oxidation rate, 30 mg/d
reduced lymphocyte DNA damage and urinary 8-OHdG in
healthy subjects [187]. A similar result was found in diabetic
patients receiving 10 mg/d of lycopene. The supplementation
had no effect on total antioxidant capacity or oxidized LDL, but
reduced MDA in serum [188]. Supplementing 12 mg/d lycopene
for 56 days reduced H
2
O
2
-induced DNA damage in lymphocytes,
butitdidnotpreventDNAdamage[189], although supplemen-
tation with 15 mg/d lycopene for 1 week prevented H
2
O
2
-induced
DNA damage in lymphocytes [190].
A few studies have investigated carotenoid supplementa-
tion and their effect on factors involved in the NF-κB pathway.
Lycopene-rich foods have received the most attention,
possibly due to the relation to tomatoes and the Mediterra-
nean diet. For example, tomato juice administered for 2 weeks
to human subjects reduced IL-2 and IL-4 secretion of
peripheral blood mononuclear cells (PBMCs) [191], and the
consumption of a tomato-based drink for 26 days lowered
TNF-αsecretion by 34% [192]. Although these results are
promising, other studies with lycopene supplementation or
tomato products showed rather limited effects, that is, no
effect on apoptotic markers [193],MDA[194], C-reactive
protein [195], and nitrite/nitrate [194]. In early arthrosis
patients, supplementing 20 mg lutein per day for 3 months
reduced plasma IL-6 and MCP-1 and improved serum lipids,
when compared with subjects receiving a placebo. This
indirectly indicated that NF-κB may have been involved
[196]. In subjects receiving enteral nutrition, carotenoid
enrichment (3 mg/1500 kcal) reduced NF-κB lymphocyte
expression, after 3 months of intervention, as compared
with a control group [197].
In contrast, 2 or 8 servings/d of fruits and vegetables for 4
weeks did not show any effect on TNF-αand IL-12 production,
and the same was observed for C-reactive protein concentra-
tions. However, consuming more than 8 servings/d reduced
C-reactive protein concentration [198]. In overweight or obese
postmenopausal woman, consuming 2, 5, or 10 servings of
vegetables/d for 3 weeks had no effect on urinary 8-isoprostane
F2αor serum C-reactive protein [199], despite a clear increase in
carotenoid plasma levels. Perhaps the short time of interven-
tion limited observable effects on the inflammation markers.
Only a few human studies have investigated the relation-
ship of carotenoids and Nrf2 expression. A recent study by
Talvas et al [200] showed that cells incubated with sera from
men who consumed red tomato paste had a significant
up-regulation of insulin-like growth factor binding protein 3
(IGFBP3) and Bax/Bcl-2 ratio and down-regulation of cyclin-D1,
p53, and Nrf2. These are all genes implicated in the cell cycle,
cell stress response, apoptosis, or cell proliferation. Moreover,
the study showed that cells incubated with sera from men
who consumed purified lycopene had significant up-regulated
IGFBP3, c-fos, and μPAR, which are genes implicated in cell
proliferation and carcinogenesis. In another recent study,
lycopene supplementation (7 mg/d for 3 weeks) was unable to
modulate HO-1 in lymphocytes of young men, perhaps
because they were already healthy [201]. Middle-aged, mod-
erately overweight subjects who received a lycopene-rich diet
(224-350 mg) or lycopene supplements (70 mg/week) for 12
weeks showed significantly improved serum-amyloid A,
which is a marker of systemic and high-density lipoprotein
associated inflammation [202].
6. Conclusions, gaps of knowledge,
and future research
Many mechanistic (ie, in vitro studies) investigating the effect
of carotenoids on various markers of oxidative stress and
inflammation have indicated beneficial health effects, by
influencing transcription factors, such as NF-κB or Nrf2, and
their downstream targets, such as IL-8 and PGE2 or HO-1, SOD,
respectively. However, other, less well-studied molecular
targets, such as RAR, may also play a role.
Thus, although several pathways possibly related to
carotenoids, inflammation, and oxidative stress have been
16 NUTRITION RESEARCH XX (2014) XXXXXX
Please cite this article as: Kaulmann A, Bohn T, Carotenoids, inflammation, and oxidative stressimplications of cellular
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uncovered, many aspects remain poorly understood and
require more research. This research should include the
synergistic aspects between different carotenoids and other
compounds such as vitamins, their dosing, and the role of
carotenoid breakdown products or metabolites. Taking carot-
enoid supplements alone has often failed or even indicated
negative effects on disease risk in some trials, especially in
subjects at risk for oxidative stress, such as smokers. Although
this remains controversial, it may be explained by the absence
of synergistic effects with compounds normally present in
whole foods, such as other antioxidants like vitamin E or C,
[203,204]. Carotenoids, when taken as a supplement and not in
a food matrix, especially when taken in significant doses, may
act as prooxidants, which would be in line with the effects
seen in vitro [205].
Future research should investigate which carotenoids and
their metabolites, such as apocarotenoids or water-soluble
derivatives, may constitute suitable modulators to alter
pathways related to oxidative stress and inflammation.
Research should also focus on the effects that other matrix
components may have on their metabolism and potential
anti-inflammatory properties. Such studies will likely ad-
vance understanding into the potential dose-relation effects.
Further, studying of molecular targets of this promising group
of secondary plant compounds are warranted.
Acknowledgment
This study was supported by the Fonds National de la
Recherche Luxembourg (C10/SR/819345). Part of images from
Motifolio drawing toolkits (www.motifolio.com) were used in
the figure preparation.
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Please cite this article as: Kaulmann A, Bohn T, Carotenoids, inflammation, and oxidative stressimplications of cellular
signaling pathways and relation to chronic disease preven..., Nutr Res (2014), http://dx.doi.org/10.1016/j.nutres.2014.07.010