International Journal of Radiation Biology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/irab20

Effect of ultraviolet radiation on the Nrf2 signalling

pathway in skin cells

Alena Ryšavá, Jitka Vostálová & Alena Rajnochová Svobodová

To cite this article: Alena Ryšavá, Jitka Vostálová & Alena Rajnochová Svobodová (2021): Effect
of ultraviolet radiation on the Nrf2 signalling pathway in skin cells, International Journal of Radiation
Biology, DOI: 10.1080/09553002.2021.1962566

To link to this article: https://doi.org/10.1080/09553002.2021.1962566

Accepted author version posted online: 31

Jul 2021.

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Effect of ultraviolet radiation on the Nrf2 signalling pathway in skin cells

Alena Ryšavá , Jitka Vostálová, Alena Rajnochová Svobodová *

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry,

Palacký University, Olomouc rysava.alena@hotmail.com

* Corresponding author:

Alena Rajnochová Svobodová

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Department of Medical Chemistry and Biochemistry

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Palacký University

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Hněvotínská 3, 775 15 Olomouc

Czech Republic
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Tel: +420585632316
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Fax: +420585632302

E-Mail: alf.svoboda@seznam.cz
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Abstract

Purpose: Excessive exposure of skin to solar radiation is associated with greatly increased
production of reactive oxygen and nitrogen species (ROS, RNS) resulting in oxidative stress
(OS), inflammation, immunosuppression, the production of matrix-metalloproteases, DNA
damage and mutations. These events lead to increased incidence of various skin disorders
including photoageing and both non-melanoma and melanoma skin cancers. The ultraviolet
(UV) part of sunlight, in particular, is responsible for structural and cellular changes across
the different layers of the skin. Among other effects, UV photons stimulate oxidative damage
to biomolecules via the generation of unstable and highly reactive compounds. In response to
oxidative damage, cytoprotective pathways are triggered. One of these is the pathway driven

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by the nuclear factor erythroid-2 related factor 2 (Nrf2). This transcription factor translocates

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to the nucleus and drives the expression of numerous genes, among them various detoxifying

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and antioxidant enzymes. Several studies concerning the effects of UV radiation on Nrf2
activation have been published, but different UV wavelengths, skin cells or tissues and

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incubation periods were used in the experiments that complicate the evaluation of UV
radiation effects.
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Conclusions: This review summarizes the effects of UVB (280–315 nm) and UVA (315–
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400 nm) radiation on the Nrf2 signalling pathway in dermal fibroblasts and epidermal
keratinocytes and melanocytes. The effects of natural compounds (pure compounds or
mixtures) on Nrf2 activity and level as well as on Nrf2-driven genes in UV irradiated human
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skin fibroblasts, keratinocytes and melanocytes are briefly mentioned as well.

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Keywords
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Nrf2; skin; UV radiation; oxidative stress; photoprotection; phytochemicals

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Highlights

UVB radiation is a rather poor activator of the Nrf2-driven pathway in fibroblasts

UVA radiation stimulates Nrf2 activation in dermal fibroblasts
Effects of UVA on the Nrf2 pathway in keratinocytes and melanocytes remain unclear
Long-term Nrf2 activation in keratinocytes disturbs their normal differentiation
Pharmacological activation of Nrf2 in skin needs to be performed carefully
Introduction
Skin covers our body and, among many other important functions, provides protection
against environmental stressors including mechanical, physical, biological, and chemical
hazards (Ali and Yosipovitch 2013). For this purpose, skin has developed an efficient sensing
and counteracting system that allows it to respond to environmental changes. Skin tissue is
able to produce a variety of biologically active compounds, such as hormones and
neurotransmitters that work directly in the skin as well as in other tissues reviewed by
(Slominski and Wortsman 2000; Slominski et al. 2012b). Solar radiation represents one of the
most important environmental factors that influences skin physiology and pathology

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(Slominski et al. 2018). Exposure to the sun is important for endogenous synthesis of

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melanin, a pigment that colours the skin and is important in protecting against solar radiation

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(Del Bino S et al. 2018; Svobodová and Vostálová 2010). Sunlight is also essential for
vitamin D production (Holick 2016; Bikle 2014; Slominski et al. 2020a), necessary for

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calcium metabolism (McLafferty et al. 2012) and the immune system (Schwalfenberg 2011).
On the other hand, solar radiation stimulates damage to skin cells and structures. These
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injuries range from erythema and sunburn to premature skin ageing and carcinoma. Due to
changing lifestyles and the depletion of the ozone layer, our skin is exposed to higher amounts
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of solar rays, with the occurrence of solar light-associated skin disorders trending upwards.
However, skin cells are equipped with several protective mechanisms that diminish the
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negative effects of solar radiation (Svobodová and Vostálová 2010). One such mechanism is
the activation of the Nrf2 signalling pathway controlling cellular antioxidant homeostasis.
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Therefore, solar radiation, by inducing OS, modulates the Nrf2 signalling pathway and thus
improves protection against solar radiation. This review focuses on the effect of ultraviolet
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(UV) radiation on Nrf2 activation/activity in skin cells, particularly keratinocytes,

melanocytes and fibroblasts. The potential of natural compounds/extracts to modulate the
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Nrf2 signalling pathway in UVA irradiated skin cells is also briefly summarized.

Skin Structure and Cells

Skin is the largest human organ with an area, weight and thickness of approximately 1.5-
2.0 m2, 3.0-4.5 kg and 0.1-4.0 mm, respectively (Kanitakis 2002). Skin tissue is a poorly
water-permeable barrier with a slightly acidic hydrophilic film of pH 4-6. It consists of three
layers: epidermis, dermis and hypodermis (Ali and Yosipovitch 2013). The outermost layer,
the epidermis, is an epithelial self-renewing layer of ectodermal origin whose surface is in
direct contact with the environment. It is separated from the adjacent dermis by the basement
membrane. The dermis is a layer of mesenchymal connective tissue rich in collagen, elastin
and various cell types. The hypodermis, the lowermost layer, is derived from the mesoderm
and is largely composed of adipose cells and macrophages (McLafferty et al. 2012).
The main cell type in the epidermis (around 80 %) are cubical cells called keratinocytes.
Other cells include melanocytes (responsible for pigment synthesis), Langerhans cells
(responsible for immunological reactions) and Merkel cells with receptor functions.
Keratinocytes form several layers of variously differentiated and stratified cells. Each layer is
characterised by specific functions as well as the expression of proteins such as keratins.

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While the basal layer of keratinocytes is responsible for the production of new keratinocytes,

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the outermost layer of the epidermis, the stratum corneum, consists of several layers

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(normally around 15) of completely keratinised dead cells (corneocytes) and an extracellular
lipid matrix (Fig. 1) (Mescher 2016). Corneocytes, terminally differentiated keratinocytes,

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provide mechanical reinforcement, protect underlying mitotically active cells from UV
damage, regulate cytokine-mediated inflammation initiation, and maintain hydration. The
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extracellular lipid matrix that creates the brick-and-mortar organization of the stratum
corneum regulates permeability, initiates corneocyte desquamation, has antimicrobial activity,
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excludes toxins, and allows for selective chemical absorption (Murphrey and Zito 2020).
Overall, keratinocytes represent the first line of the body’s defence against environmental
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attack. Their strong resistance to external hazards and quick response is related to the
presence of specific receptors for keratinocyte growth factor (KGF), localized on the
keratinocyte surface. KGF mediates a signal for proliferation where epidermal damage has
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occurred and stimulates rapid cell renewal (Braun et al. 2002; Gęgotek and Skrzydlewska
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2015).
Melanocytes are the second most abundant epidermal element (1-2 %), situated in the basal
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layer and with an origin in neural crests. They are small, rounded cells with numerous long
cytoplasmic appendixes that penetrate the epidermal stratum basale and stratum spinosum
(Fig. 1) (Kanitakis 2002; Mescher 2016). Melanocytes synthesize the melanin pigments, in
particular eumelanin, pheomelanin, and mixed melanin, which participate in skin colouring
and represent one protection mechanism against solar radiation. Synthesis of melanin arises
from L-tyrosine and requires the enzyme tyrosinase (Slominski et al. 2004; Slominski et al.
2012a). Melanin has the ability to scatter UV rays and absorb UV, visible and infrared
radiation as well as scavenge reactive oxygen and nitrogen species (ROS, RNS) that have
been produced. However, melanin synthesis itself generates ROS, especially the synthesis of
lighter variant pheomelanin (Smit et al. 2008). Moreover, pheomelanin can undergo
photosensitization that results in the generation of superoxide radicals, hydroxyl radicals and
hydrogen peroxide (Slominski et al. 2004). From this point of view, pheomelanin also has
pro-oxidative properties and can participate in photoageing and carcinogenic processes (see
below). In addition to melanin synthesis, melanocytes secrete a wide range of signal
molecules such as cytokines (tumour necrosis factor-α (TNF-α), interleukins), catecholamines
and nitric oxide in response to a number of stimuli (including UV radiation) and, via these
molecules, influence other epidermal and dermal cells (Svobodová and Vostálová 2010).
Merkel cells also occur in the basal layer and adhere to keratinocytes with desmosomes.

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Merkel cells are mechanoreceptors that have a dense core containing neurosecretory granules

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in the cytoplasm (Kanitakis 2002). Langerhans cells (LCs) are located in the middle region of
the epidermis (stratum spinosum). LCs originate from CD34+ cells in bone marrow and, as

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they are antigen-presenting cells, represent a major component of adaptive immunity in skin.

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LCs have dendritic projections that extend between the keratinocytes of the entire layer
(Fig. 1). The mobilization of LCs is supported by TNF-α (Bhushan et al. 2002).
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The dermis consists primarily of dense, irregular connective tissue within a matrix of
collagen and elastin fibres, which make it compressible and elastic. Fibroblasts, spindle-
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shaped cells with a well-developed rough endoplasmic reticulum, are the predominant cells in
the dermis. Fibroblasts synthesize all dermal fibres and components of the extracellular matrix
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(Fig. 1) (Mescher 2016). Other cell types in the dermis include mast cells, macrophages,
adipocytes and plasma cells. It is interwoven with nerve fibres and blood and lymphatic
vessels that also supply nutrients to the epidermis (Svobodová and Vostálová 2010).
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The subcutaneous hypodermis tissue is the deepest layer and consists of loose connective
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tissue and elastin. The main cell types are adipocytes, large, rounded elements with a lipid-
laden cytoplasm. Adipocytes are arranged in lobules that are separated by connective tissue
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containing fibroblasts, dendrocytes and mast cells. The hypodermis also contains nerve fibres
and blood vessels. The layer is loosely connected to bone and muscles which allows the skin
to move freely. The hypodermis protects from mechanical injuries and contributes to thermal
insulation and energy storage (Kanitakis 2002).

Solar Radiation
Solar radiation is a general term for the electromagnetic radiation emitted by the sun and
can be divided into different spectral regions, such as UV, visible and infrared. Recent
research has shown that all solar radiation spectral regions have adverse biological effects on
living organisms. However, UV wavelengths have been the most intensively studied and are
still recognized as being the most aggressive towards cells and tissues. UV wavelengths are
further subdivided into UVA (400-320 nm), UVB (320-280 nm) and UVC (100-280 nm). The
toxicity of UV increases the shorter the wavelength (Ikehata and Yamamoto 2018).
Due to having the highest energy, UVC radiation has the greatest potential to damage cells,
even after short exposure times. It is highly mutagenic as the absorption maximum of DNA is
within the UVC waveband (260-265 nm). Fortunately, UVC photons are completely absorbed
in the stratospheric ozone layer, together with most UVB photons (100-295 nm) (Azami et al.
2019; Ikehata and Yamamoto 2018; Svobodová and Vostálová 2010). Nevertheless, 5-10 %
of the UV radiation that reaches the Earth’s surface is UVB. Due to ozone layer depletion, the

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amount of UVB photons reaching the surface of the planet is increasing (Young 2006). UVB

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is mostly absorbed by the epidermis, the majority (~ 70 %) in the stratum corneum, which is
formed from corneocytes (Kulms et al. 2002; Svobodová and Vostálová 2010). UVA

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represents up to 95 % of the UV rays that reach the Earth’s surface, with no considerable
absorption occurring in the ozone layer. UVA radiation is divided into two parts: wavebands
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UVA1 (400-340 nm) and UVA2 (340-320 nm). Around 80 % of UVA reaches the dermo-
epidermal junction and penetrates deeper into the papillary dermis. About 10 % of UVA even
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manages to reach the hypodermis (Pittayapruek et al. 2016; York and Jacobe 2010). The
amount of solar UV radiation reaching the Earth's surface, and the UVA/UVB ratio, depends
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on a number of factors, such as latitude, altitude, season, cloud cover, time of day, and time of
year (Young 2006).
UV photons provoke direct or indirect cellular damage; the mechanism depends on
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wavelength and the type of chromophore. UVB (as well as UVC) photons are absorbed by a
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variety of molecules such as pyrimidines; purines and their derivatives; aromatic amino acids,
peptides and proteins containing the amino acids; melanin monomers, polymers and
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precursors; quinones, trans-urocanic acid, melatonin, indoles; 7-dehydrocholesterol or

unsaturated fatty acids and compounds containing them (Slominski et al. 2018). The most
hazardous is energy absorption by DNA, which stimulates the formation of DNA lesions,
most frequently cyclobutane pyrimidine dimers (CPDs). The second most abundant lesions
are pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs), which are more rapidly repaired
than CPDs (Cadet et al. 2015). UVB photons also stimulate the modification of amino acids
with, primarily, indolyl radicals from tryptophan and phenoxyl radicals from tyrosine
(Hawkins and Davies 2019), which alter protein function and cellular signalling. Therefore, it
is mainly UVB that significantly contributes to skin cancer development and premature skin
ageing (Pittayapruek et al. 2016). In contrast to UVB, UVA photons are weakly absorbed by
DNA and attack other chromophores, mainly heme, NADH, NADPH, quinones, flavins and
porphyrins. Through these chromophores, UVA photons are much more efficient in inducing
oxidatively generated damage by greatly increasing the production of ROS and RNS (Cadet et
al. 2012; Slominski et al. 2018). However, there is evidence that UVA photons are also able
to induce formation of CPDs, either by direct excitation or via photosensitization. The
frequency of CPD production is much higher than that of 8-oxo-7,8-dihydro-2´-
deoxyguanosine (8-oxo-dGuo), an oxidatively modified DNA base (Cadet et al. 2015).
Moreover, in normal melanocytes and keratinocytes, that receive melanin from melanocytes,
“dark CPDs” are also produced. These lesions develop for several hours after the end of UV

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exposure (in the absence of UV photons) and constitute the majority of all CPDs. Production
of these “dark CPDs” is associated with melanin, mainly pheomelanin excited by UV-induced

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ROS/RNS. The energy is transferred to DNA bases and induces CPDs (Premi et al. 2015).

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ROS and RNS, which can also be produced in lesser amount by UVB photons, oxidize
cellular biomolecules such as lipids, proteins and DNA resulting in the production of
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structurally varied oxidative products (lipid hydroperoxides, protein carbonyls, DNA-protein
crosslinks, single strand breaks or oxidatively modified bases) (Svobodová and Vostálová
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2010). Even though mutations caused by UVA photons occur with 1000-times lower
efficiency than UVB ones, due to a less pronounced DNA protective response (expression of
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tumour suppressor p53), the elimination of lesions is lower and slower and thus the UVA-
caused lesions may have a more mutagenic outcome (Kappes et al. 2006). Therefore, UVA
radiation enhances the mutagenic impact of UVB by causing oxidative damage to the
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proteome, including DNA repair enzymes, thus impairing the protective, anti-mutagenic and
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reparative responses of the damaged cells and thus promoting the carcinogenetic effect of
UVB (Calzavara‐Pinton et al. 2019). On the other hand, ROS/RNS, oxidised products and
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DNA lesions stimulate defensive and healing processes that include the expression of several
signalling molecules, such as cytokines, chemokines, protein kinases (e.g. mitogen-activated
protein kinases (MAPK)), transcription factors (e.g. NF-E2-related factor 2 (Nrf2), nuclear
factor kappa B (NFB), and activator protein-1 (AP-1)), leading to the activation of specific
signalling pathways (López-Camarillo et al. 2012). These events are directed at strengthening
protective mechanisms, the repair and/or elimination of damaged molecules or the induction
of cell death (apoptosis, necrosis). The key factor in redox-sensitive conditions has been
recognized as Nrf2.
Nuclear Factor Erythroid-2 related Factor 2 (Nrf2)
To counteract the detrimental effects of exogenous damage, mammalian cells have evolved
a hierarchy of sophisticated sensing and signalling mechanisms. One of the major protective
cellular responses is activation of the Nrf2 transcription factor (also known as nuclear factor
erythroid-derived 2-like 2). Nrf2 is coded by the gene NFE2L2 and is a member of the
“Cap’n’Collar” (CNC) subfamily of basic leucine zipper (bZIP) transcription factors, which
also includes the related Nrf1 and Nrf3 proteins, as well as p45 nuclear factor erythroid-
derived 2 (p45 NF-E2), transcription factor BTB domain and CNC homolog 1 (Bach1) and 2
(Bach2) (Canning et al. 2015). These proteins require other leucine zipper proteins, such as

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small musculo-aponeurotic fibrosarcoma proteins (sMaf), Jun proteins, or c-Fos to function.

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Upon hetero-dimerization with these interaction partners, Nrf2 binds to DNA with the
characteristic sequence 5′-TGACNNNGCA-3′ (where N is any nucleotide) in the promoters

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of target genes, called the antioxidant response element (ARE) or electrophile response

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element (EpRE) (Braun et al. 2002). Binding of the Nrf2 heterodimer to the ARE sequence
results in the transcriptional activation of a number of genes. It was reported that Nrf2 has
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more than 500 target genes including, for example, proteins essential for redox balance,
cytoprotection, phase II detoxification, carbohydrate, lipid and heme/iron metabolism,
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transporters, growth factors, and cell survival (Zhao et al. 2021). Antioxidant enzymes and
detoxifying proteins comprise NAD(P)H quinone oxido-reductase (NQO1), epoxide
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hydrolase 1 (EPHX1), carbonyl reductase 1 (CBR1), UDP-glucuronosyltransferases (UGT),

several ATP-binding cassette transporters (ABC transporters), glutathione (GSH) synthesizing
enzymes (glutamate-cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM)),
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GSH S-transferases (GST), GSH reductase (GSR), GSH peroxidases (GPX), thioredoxin
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reductase (TrxR), thioredoxins (TRX), peroxiredoxins (PRX), heme oxygenase 1 (HO-1) and
other cytoprotective proteins including itself (Nrf2) and its inhibitors Kelch-like ECH-
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associated protein 1 (Keap1) or p62 (Hayes and Dinkova-Kostova 2014; Sánchez-Martín and
Komatsu 2018). The expression of genes encoding catalase (CAT) and superoxide dismutase
1 (SOD1) seems to also be under Nrf2 control, as suggested by experiments on Nrf2 knockout
mice (Chan and Kan 1999). Studies also document that Nrf2 contributes to the anti-
inflammatory process by regulating inflammatory cell recruitment, stimulating anti-
inflammatory gene expression (e.g. interleukin-10 (IL-10)) and inhibiting pro-inflammatory
gene progression (e.g., interleukin-6 (IL-6) and interleukin-1β (IL-1β)) (Ahmed et al. 2017).
The pro-inflammatory cytokine TNF-α was further demonstrated to have a bimodal effect on
the Nrf2 signalling pathway. While its intense inflammatory activation suppressed the
expression of antioxidant proteins such as GPX, GCLM, GCLC, SOD1, CAT, NQO1 and the
level of GSH, a low TNF-α level had the opposite effect and appears to be a protective agent
(Shanmugam et al. 2016).
The Nrf2 protein in humans is 605 amino acids long and contains seven highly conserved
regions known as Nrf2-ECH homology (Neh) domains, see Fig. 2. Neh1 contains the CNC-
bZIP region, which is necessary for DNA binding and also mediates hetero-dimerization with
its transcriptional partners, mainly sMaf. The Neh2 domain has been identified as the negative
(29)
regulatory domain and specifically binds to the Nrf2 inhibitor Keap1 through DLG and
(79)
ETGE motifs (Canning et al. 2015). The Neh2 domain also contains seven lysine residues

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essential for the ubiquitination and subsequent proteasomal degradation of Nrf2. The Neh3,

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Neh4 and Neh5 domains play a part in the activation of Nrf2-driven gene transcription. The

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Neh6 domain is a serine rich area involved in negative regulation of Nrf2 stability,
(343) (382)
independent of Keap1. The domain contains DSGIS and DSAPGS motifs that are

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recognized by -transducing repeat-containing protein (-TrCP). The Neh7 domain
participates in the repression of Nrf2 transcriptional activity by the retinoid X receptor 
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(RXR-α) through a physical association between the two proteins (Tonelli et al. 2018). As
shown in a detailed analysis, Nrf2 activity is tightly controlled by positive and negative
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factors (proteins) and can be regulated through Nrf2 stability, post-transcriptional

modifications and the availability of binding partners reviewed by (Tonelli et al. 2018).
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Regulation of Nrf2 Activity

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After decades of intensive study, it is evident that Nrf2 is strictly controlled and under
normal homeostatic conditions Nrf2 is kept low by continuous proteasomal degradation.
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Keap1 (sometimes referred to as an inhibitor of Nrf2 with the abbreviation INrf2) was the first
identified Nrf2 binding partner and regulatory protein of Nrf2. Keap1 was named so due to its
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structural similarities to the Drosophila Kelch protein (Dinkova-Kostova et al. 2017). Keap1
is a 624-amino acid cysteine-rich cytosolic protein that belongs to the BTB-Kelch family of
proteins. Human Keap1 contains 27 cysteine residues, distributed throughout the protein. The
presence of many cysteines causes Keap1 to have a high redox sensitivity and vulnerability to
oxidation or to covalent modification by electrophiles and thus the cysteine residues serve as
the sensors for Nrf2 inducers. Besides exogenous stressors, there are several endogenous
Keap1 modifiers such as nitric oxide, fumarate, hydrogen peroxide, cyclopentenone
prostaglandins and nitro-fatty acids that can inactivate Keap-1 (Hayes and Dinkova-Kostova
2014). Only some of the cysteine residues in Keap1 were, however, identified as critical for
Nrf2 activation (Dinkova-Kostova et al. 2017). The cysteines Cys273 and Cys288 seem to be
required to control Nrf2 under both basal and stress conditions while Cys151 is essential
under stress conditions (electrophile interaction). Other Keap1 cysteines, including Cys226,
Cys434, and Cys613, seem important for interactions with specific electrophilic inducers
(Canning et al. 2015). Keap1 forms a functional E3 ubiquitin ligase complex by interacting
with Cullin-3 (Cul3) and GST-tagged Roc1 (Rbx1) protein. In the absence of OS (basal state),
the cellular level of Nrf2 is kept lower than the levels of Keap1 and Cul3 proteins (Iso et al.
2016), which allows for the basal activity of Nrf2 and maintains the housekeeping expression
of many related antioxidant and detoxifying genes. In this state, Nrf2 binds to the Keap1-

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Cul3-Rbx1 complex tethered to the actin cytoskeleton. This complex suppresses the Nrf2

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function passively by sequestering Nrf2 in the cytoplasm, as well as actively by
(29)

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polyubiquitination at multiple lysine residues in the Neh2 domain of Nrf2 (between DLG
and (79)ETGE motifs), thus facilitating Nrf2 degradation by the 26S proteasome (Sykiotis and

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Bohmann 2010). Keap1 is thereafter regenerated and prepared for binding newly synthetized
Nrf2 (Hayes and Dinkova-Kostova 2014).
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Low Nrf2 level under normal conditions is also maintained Keap-1-independently. Some
MAPKs, in particular p38, have been shown to phosphorylate Nrf2 and promote the
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association between Nrf2 and Keap1 proteins, thereby inhibiting the nuclear translocation of
Nrf2 and transcriptional activity (Fig. 2) (Boo 2020). Stimulation of Nrf2 ubiquitination and
(343) (382)
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degradation can occur via the interaction of the DSGIS and DSAPGS motifs of Nrf2
with -TrCP and following Nrf2 phosphorylation by a serine/threonine kinase called glycogen
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synthase kinase-3 (GSK-3) at the Neh 6 domain, particularly at Ser344 and Ser347 in humans
(and at Ser335 and Ser338 in mice) (Boo 2020; Hayes and Dinkova-Kostova 2014). GSK-3
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may also alter Nrf2 activity indirectly via Fyn kinase (FynK) activation (see below) (Hayes
and Dinkova-Kostova 2014). Constitutively active GSK-3 may be inhibited via
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phosphorylation by several kinases such as protein kinase B (PKB/Akt), PKC,

phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) that
potentially enhance Nrf2 activity (Jope et al. 2007; Tonelli et al. 2018).
Under OS, due to the presence of electrophilic chemicals or oxidants, Nrf2 is activated.
This activation occurs predominantly through modification of Keap1. Electrophiles react with
Keap1 via Michael addition and form covalent bonds with the cysteine residues. Oxidants can
also cause oxidation of cysteine residues of Keap1 or stimulate activity of some kinases and
thus cause the release of Nrf2 from Keap1 in the cytosol, and translocation of Nrf2 into the
nucleus. Activation of Nrf2 can further occur via phosphorylation at serine and/or threonine
residues. Several protein kinases have been identified that cause activating phosphorylation of
Nrf2 (Fig. 2). Protein kinase C (PKC) was found to phosphorylate the Neh2 domain of Nrf2 at
Ser40 (Nrf2-PSer40) (Huang et al. 2002), protein kinase R-like endoplasmic reticulum kinase
(PERK) the Neh2 domain at Thr80 (Osama et al. 2020), adenosine monophosphate (AMP)-
activated protein kinase (AMPK) the Neh1 domain at Ser558 in humans (and Ser550 in mice),
casein kinase 2 (CK2) the Neh4 and Neh5 domain at multiple sites (Boo 2020) and ERK and
c-Jun N-terminal kinase (JNK), belonging to the MAPK family, at several serine residues
(Ser215, Ser408, Ser558, Thr559, Ser577 of Nrf2 were proposed as the potential targets) (Boo
2020). In relation to the topic being discussed, PERK, AMPK, ERK, p38 and JNK are

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activated by UVA radiation (Lee et al. 2010) as well as UVB radiation (Ji et al. 2017). The

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reactions result in Nrf2 conformation changes and dissociation from the complex with Keap1

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(Fig. 2). Free Nrf2 cannot be ubiquitinated and degraded. In turn, Nrf2 is stabilized,
translocated to the nucleus where it forms a hetero-complex with other leucine zipper

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proteins, interacts with ARE and activates the target genes that ensure cell survival (Fig. 3)
(Gęgotek and Skrzydlewska 2015; Chun et al. 2014; Yang et al. 2021). Some papers suggest
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that modification of Keap1 by electrophiles does not result in Nrf2 release, but the Nrf2-
Keap1 complex is instead driven to proteasomal degradation and newly synthetized Nrf2
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directly translocates to the nucleus (Hayes and Dinkova-Kostova 2014). Besides modification
of cysteine residues, Keap1-Nrf2 interaction may be disrupted by the protein p62, also known
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as sequestosome-1 (Osama et al. 2020). Normally, p62 works as a protein adaptor that binds
to ubiquitylated protein aggregates, damaged mitochondria, etc. and mediates their delivery to
the autophagosomes (Jiang et al. 2015). p62, through its Keap-interacting region (KIR, AA
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346-359), is able to interact with the Kelch domain in Keap1, in a similar manner to the
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interaction of Keap1 with Nrf2. Specifically, the interaction involves Asp349, Pro350,
Ser351, Thr352, Glu354 and Leu355 residues in p62 and Tyr334, Ser363, Arg380, Asp382,
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Arg415, Gln530, Ser555 and Ser602 in Keap1. After p62 phosphorylation (p-p62) by the
mechanistic targeting of rapamycin complex 1 (mTORC1) or transforming growth factor-β-
activated kinase 1 (TAK1) at Ser351, p-p62 binding affinity for Keap1 significantly increases
due to an additional interaction between Ser351 in p-p62 and Arg483 and Ser508 in Keap1,
thus p-p62 competes with the ETGE motif of Nrf2 for Keap1 binding (Silva-Islas and
Maldonado 2018). Consequently, the Keap1-p-p62 complex undergoes autophagy-dependent
degradation. Nrf2 is stabilized, translocated into the nucleus and induces target genes (Fig. 3)
(Ichimura et al. 2013). Interestingly, p62 belongs to the group of genes that are positively
regulated by Nrf2. Therefore, if autophagy is disrupted or lost, p62 accumulates and interacts
with Keap1, resulting in constitutive Nrf2 activation and overexpression of p62 and other
Nrf2 target genes, causing severe tissue damage (Komatsu et al. 2010). Among others, p62
contributes to the growth of tumour cells and increased p62 levels have been found in several
human cancers, including melanoma and skin squamous cell carcinoma. The p62 protein
stimulates resistance to chemotherapeutic drugs as well (Silva-Islas and Maldonado 2018).
p62 level is modified by UV radiation. While UVA radiation stimulates p62 transcription in
skin cells via activation of transcription factor EB (TFEB), a regulator of autophagy and
lysosomal gene expression (Sample et al. 2017; Zhao et al. 2013), UVB radiation reduces p62
level (Lorca and Wu 2020; Yang et al. 2012). From this point of view, UVA can contribute to

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skin tumorigenesis and tumour progression.

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In addition to Keap1, other regulatory proteins exist that also influence Nrf2 activity:

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Bach1, Fyn kinase (FynK), caveolin-1 (Cav-1) and p21 (Fig. 3), at least, have been described.
Bach1, a member of the CNC family transcription factors, is a nuclear protein that makes

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heterodimers with sMaf (sMafK and sMafG) and thus blocks the activity of Nrf2. This has
been described for, at least, HO-1 (Suzuki et al. 2004) and NQO1 (Kaspar and Jaiswal 2010)
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gene expression. Some agents, such as the heme that is released under OS from heme-
containing proteins (Igarashi and Sun 2006; Suzuki et al. 2004; Suzuki et al. 2003), as well as
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cadmium (Suzuki et al. 2003), induce the removal of Bach1 from the ARE-related sMaf-
associated recognition element that activates ARE-driven genes. This Bach1 release is
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followed by its nuclear export to the cytosol, dependent on chromosomal region maintenance-
1 protein (Crm1; exportin-1) and extracellular signal-regulated kinase-1/2 (ERK1/2) activity.
An antioxidant tert-butylhydroquinone activates unknown tyrosine kinase(s) that
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phosphorylate(s) Bach1 at Tyr486, which also leads to nuclear export, ubiquitination and
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degradation by the proteasome system (Fig. 3) (Kaspar and Jaiswal 2010).

FynK has been identified as a negative regulator of Nrf2. FynK belongs to the Src family
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of tyrosine kinases and, compared to other family members, is more ubiquitously expressed
(Courtneidge et al. 1993). After FynK phosphorylation at threonine residue(s) (FynK-PThr) by
activated GSK-3, FynK-PThr translocates and accumulates in the nucleus. FynK-PThr then
phosphorylates Nrf2 at Tyr568 (Nrf2-PTyr568) in mice (Jain and Jaiswal 2007) or Tyr576
(Nrf2-PTyr576) in humans (Hayes and Dinkova-Kostova 2014), resulting in the nuclear export
of Nrf2-PTyr568 via exportin 1 to the cytosol and its proteasomal degradation. As an early
response to OS, FynK-PThr present in the nucleus is phosphorylated by an unknown kinase at
Tyr213 (FynK-PThr-PTyr213) and is then exportin-1-dependently exported from the nucleus and
is degraded. This removal of diphosphorylated FynK (inactive FynK) allows the subsequent
binding of free Nrf2 (newly transported from the cytosol to the nucleus upon OS) to ARE and
the expression of targeted genes. After activation of the Nrf2-driven cytoprotective genes,
FynK-PThr is imported to the nucleus and stimulates Nrf2 phosphorylation (Nrf2-PTyr576), its
nuclear removal via exportin 1 and proteasomal degradation (Fig. 3) (Ishizawa et al. 2015;
Kaspar and Jaiswal 2011).
Caveolins are the structural and scaffolding proteins of caveolae, small (50- to 100-nm)
omega-shaped invaginations of the cell surface plasma membrane, rich in glycosphingolipids
and cholesterol. Caveolae occur in a variety of cell types, including fibroblasts, epithelial and
endothelial cells (Cohen et al. 2004; Volonte et al. 2013). There are three isoforms of

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caveolins (1-3). Cav-1 is the principal component of caveolae, with an unusual hairpin-like

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conformation that participates in vesicular trafficking and signal transduction events. It is

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expressed ubiquitously and Cav-1-enriched cellular organelles include the mitochondrion, the
nucleus, the Golgi complex, and the endoplasmic reticulum (Wang et al. 2017). As has been

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documented, Cav-1 is an OS-related protein, as OS modulates the expression,
posttranslational modification and degradation of Cav-1. Under basal conditions, Cav-1
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constitutively interacts with Nrf2 in both the cytosol and nucleus. Under OS, ROS/RNS
decrease Cav-1 expression, induce its proteasomal degradation and phosphorylation and also
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inhibit its palmitoylation, which decreases Cav-1 membrane association. These events result
in reduced Cav-1-Nrf2 interaction, reviewed recently by (Wang et al. 2017) (Fig. 3). In
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addition, Cav-1 knockdown affects the interaction between Nrf2 and Keap1 and increases
Nrf2 transcription activity. Mutation of the Cav-1 binding motif on Nrf2 effectively attenuates
their interaction and results in higher transcription activity of Nrf2-targeted genes. It also
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induces higher levels of antioxidant enzymes compared to a wild-type control (Li et al. 2012).
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The p21 protein regulates many cellular processes including cell cycle arrest, apoptosis and
DNA replication and repair. Under OS, p21 is up-regulated and involved in cell survival
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(Silva-Islas and Maldonado 2018). This occurs via posttranslational activation of Nrf2 by
(29)
competition with Keap1 for Nrf2 binding. p21 is able to interact with the DLG and
(79) (154)
ETGE motifs in Nrf2 through the KRR sequence at the C-terminal end in its structure.
This interaction does not cause dissociation of the Keap1-Nrf2 complex. However, it prevents
Nrf2 ubiquitination and increases Nrf2 stabilization (Chen et al. 2009). Through the p21
protein, some Nrf2 inducers such as sulforaphane, diallyl disulfide and tert-
butylhydroquinone also work (Silva-Islas and Maldonado 2018). Nrf2 activation via p21
prevents skin carcinogenesis and the inflammatory response. However, this mechanism was
also found to stimulate cancer cell proliferation and increase the drug chemoresistance of
cancer cells. This supports the Nrf2 dual role theory and the role of Nrf2 in carcinogenic
processes where, in the early stages, Nrf2 is able to protect cells against the carcinogenic
process through the decrease of ROS levels, whereas over a longer time, Nrf2 promotes
cancer cell proliferation, survival and drug chemoresistance (Silva-Islas and Maldonado
2018). The level of p21 is influenced by UV radiation through the p53 protein. Low UV doses
causing mild DNA damage and stimulating a moderate increase in p53 provoke an increase in
p21 level to trigger cell cycle arrest, allowing the repair of DNA lesions. High UV doses
causing severe DNA damage stimulate a high increase in p53 that provokes expression of the
pro-apoptotic genes, including damaged-DNA binding protein 2 (DDB2). DDB2 promotes

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p21 degradation and thus stimulates apoptosis (Li et al. 2013).

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Nrf2 in Skin
As documented in many studies, Nrf2 is important for skin homeostasis. All human skin

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cells express Nrf2, however the basal level of the Nrf2 gene differs among individual cells
(Ikehata and Yamamoto 2018; Kokot et al. 2009). In keratinocytes, basal Nrf2 gene
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expression is about two-fold higher compared to fibroblasts and melanocytes (Ikehata and
Yamamoto 2018). In the epidermis, Nrf2 was detected uniformly from the basal to granular
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layers of healthy human skin (Lee et al. 2014). The Nrf2 negative regulator Keap1, however,
was predominantly found in the basal layer and lower in the spinous layer of the epidermis
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(Fig. 4). Nrf2 nuclear translocation to the nucleus gradually increased as keratinocytes
differentiated, which clearly indicates the importance of Nrf2 activation during keratinocyte
differentiation (Lee et al. 2014). However, in the murine epidermis, a gradient of Nrf2
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expression and activity was detected. In differentiated suprabasal keratinocytes, mRNA

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expression of Nrf2 and its target genes (NQO1, GCLC, GCLM, GSR, and sulfiredoxin 1) was
significantly higher compared to non-differentiated basal cells (Schäfer et al. 2010a). This
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diverse Nrf2 activity in variously differentiated keratinocytes seems to be important in the

protection of skin against environmental stressors. As shown in UVB irradiated mice, low
Nrf2 activity in basal keratinocytes, that serve as stem cells, was found to ensure a high rate of
apoptosis, most probably to avoid the proliferation of cells with damaged DNA and thus
prevent the propagation of mutations. In contrast, in suprabasal keratinocytes, Nrf2 activity
was high and resulted in the increased expression of cytoprotective genes for cell guarding, to
maintain skin/epidermal integrity (Schäfer et al. 2010b). Nrf2 expression was also found to be
important for healing skin wounds. In this process, the Nrf2 level is regulated by KGF. In
Nrf2 knockout mice, wound healing was associated with prolonged inflammation with
increased levels of pro-inflammatory cytokines (IL-1, TNF-α), but without obvious
histological abnormalities. Normal healing progress is probably compensated for by an
increased expression of Nrf3, the related transcription factor that is also stimulated by KGF
(Braun et al. 2002). Besides epidermal and dermal cells, Nrf2 is also expressed in hypodermal
adipocytes. Initially, Nrf2 function in adipocytes was implicated in the transcriptional control
of detoxifying enzymes such as NQO1, GST, UGT and sulfotransferase, metabolizing
xenobiotics that can accumulate in adipose tissue (Yoshinari et al. 2006). Later, Nrf2 activity
was found to be essential for the regulation, formation and function of adipocytes including
lipid metabolism and adipocyte sensitivity to insulin. Nrf2 may thus represent a new

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pharmacological target for the treatment of obesity and related clinical disorders. On the other

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hand, genetic modification of Nrf2 activity (the protein itself or its regulatory proteins) may

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result in affected adipogenesis and energetic metabolism. Wang et al. complexly reviewed the
topic in a recent paper (Wang et al. 2020). High Nrf2 levels were also detected in immune

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cells such as monocytes, neutrophils, macrophages, T cells, and B cells that are not directly
resident cells in skin but are important for physiological and pathological processes occurring
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in the skin. The activation of Nrf2 in immune cells blocks inflammation by inhibiting pro-
inflammatory nuclear factor kappa B (NFκB) and/or the transcription of pro-inflammatory
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cytokine genes driven by this nuclear factor. The activation of Nrf2 in immune cells may alter
the differentiation, expansion, and survival of the cells as well as the cytokines release (He et
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al. 2020).
Disruption of normal Nrf2 activity in the epidermis and/or dermis may be associated with
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the development of various skin disorders. Yang et al. documented that overexpression of
Nrf2 provoked the up-regulation of keratin 6, 16 and 17 expressions, with subsequent
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pathological keratinocyte proliferation and differentiation, which manifests clinically as

epidermal thickening and hyperkeratosis, typically occurring in psoriatic epidermal lesions.
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Hyperactivation of Nrf2 in psoriatic skin is stimulated by several pro-inflammatory cytokines,

including interleukin 17 and 22 (Yang et al. 2017a). The hyperactivation of Nrf2 may also
result from the lower expression of Cav-1 that was found in the psoriatic skin of human
volunteers (Ma et al. 2012). A mutation in the Keap1 gene causing constitutive Nrf2
accumulation in the nucleus and the expression of target genes resulted in death within 3
weeks of birth in homozygous mice. Detailed analysis revealed hyperkeratosis in the
oesophagus and stomach, causing starvation due to nutrient obstruction and stomach
ulceration. These mice also showed massive hyperthickening of the cornified layer of the
epidermis. Keap1 deficiency resulted in changes in squamous cell differentiation markers. In
particular, levels of keratin 1, 6 and loricrin were markedly increased while the level of
involucrin was reduced (Wakabayashi et al. 2003). In mice with a long-term pharmacological
Nrf2 activation, impaired desquamation, corneocyte fragility, alterations in the epidermal lipid
barrier, inflammation and overexpression of mitogens that induced keratinocyte
hyperproliferation, epidermal thickness, and hyperkeratosis were observed (Taguchi et al.
2010). Although Nrf2 is generally considered to be a cytoprotective transcription factor and a
tumour suppressor, mutations in Nrf2 or Keap1, resulting in permanent Nrf2 activation,
correlated with increased malignancy, resistance of tumour cells to chemotherapy (e.g., 5-
fluorouracil, cisplatin, oxaliplatin, paclitaxel, doxorubicin) and radiotherapy and resistance to

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apoptosis (via upregulation of anti-apoptotic genes). Thus, impaired Nrf2 activity promotes

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tumour proliferation and metastasis resulting in a worse prognosis (Sporn and Liby 2012; Xue

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et al. 2020). Nrf2 mutations have been identified in various carcinomas, including cutaneous
squamous cell carcinomas (Kim et al. 2010). Recently, it has been shown that vitiligo

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melanocytes have impaired Nrf2-ARE signalling and decreased activation of the protective
antioxidant enzymes. In people with vitiligo, a significantly lower level of nuclear Nrf2 was
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reported compared to healthy subjects, accompanied by significantly lower HO-1 protein
expression, GSH/GSSG ratio and enhanced levels of ROS and malondialdehyde. When
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vitiligo melanocytes were treated with hydrogen peroxide, a significantly lower Nrf2 nuclear
translocation, as well as HO-1 mRNA and protein level, was detected (Jian et al. 2014).
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mRNA sequencing data of primary human epidermal melanocytes with Keap1 knockdown
demonstrated significantly up-regulated genes, among them many antioxidants such as
NQO1, HO-1, GCLM, thioredoxin, thioredoxin reductase 1 and sulfiredoxin 1. Keap1
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silencing further increased tyrosinase activity, protein and mRNA level as well as melanin
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content in the melanocytes (Kim et al. 2017). It is evident that Keap1 down-regulation in
melanocytes is important for cell proliferation and survival and thus up-regulation of Nrf2
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activity may be a potential preventive strategy for autoimmune diseases including vitiligo
(Cuadrado et al. 2018; Shin et al. 2014).
The overall data on mice and humans suggests the dual role of Nrf2. While short-term
activation is essential in cell protection against OS caused by ROS/RNS overproduction,
constitutive or long-term Nrf2 activation in the epidermis is detrimental due to an abnormal
enhancement of keratinocyte differentiation. Similarly, for carcinogenic processes, in its early
stages Nrf2 is able to protect cells against the carcinogenic process through the decrease of
ROS/RNS levels, whereas at later stages Nrf2 promotes cancer cell proliferation and drug
chemoresistance.

Nrf2 in Response to UV Radiation

As the regulator of several antioxidant and cytoprotective genes, Nrf2 transcription factor
is important for the cellular response to OS provoked by various environmental factors. As
mentioned above, UVB photons are absorbed by different chromophores than UVA.
Therefore, UVB radiation is associated mainly with direct damage to biomolecules (mainly
DNA and proteins) while UVA mostly causes indirect damage associated with massive
ROS/RNS generation and the subsequent oxidative modification of biomolecules. However,

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UVB photons also stimulate ROS production. Thus, one could suppose that both UVA and
UVB will increase Nrf2 expression and/or activity in skin cells. Nevertheless, the available

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studies on UVB and UVA radiation have reported contrary results; some showed no effect or

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down-regulation, others found increased nuclear translocation and/or expression of Nrf2 and
its target genes (see below).
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Effect of UVB
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Studies on UVB radiation tend to show no or negative effects on Nrf2 activity. Exposure of
normal murine keratinocytes to UVB (10-40 mJ/cm2) did not cause activation of Nrf2 target
genes (NQO1 and GCLC) (Durchdewald et al. 2007). In cultured primary mouse fibroblasts,
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UVB doses of 15-200 mJ/cm2 stimulated a reduction in nuclear Nrf2 translocation (Hirota et
al. 2005). Kinetic studies on normal human epidermal keratinocytes (NHEK) and normal
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human epidermal melanocytes (NHEM) showed a dramatic reduction in Nrf2 mRNA level 2
h after exposure to only 10 mJ/cm2 of UVB radiation. In NHEK, the basal level normalised
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within 24 h, whereas in NHEM it remained significantly reduced after 24 h. In human skin

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explants, the same UVB dose also reduced Nrf2 mRNA level, however the decrease was not
as strong as in NHEK and NHEM. UVB exposure also negatively affected mRNA levels of
HO-1, GST isoform Pi (GSTPi) and gamma-glutamylcysteine synthetase (-GCS), also
known as glutamate cysteine ligase (GCL), in both cell types as well as in human organ skin
cultures (Kokot et al. 2009). Similarly, a study with human neonatal melanocytes showed that
UVB radiation (30 mJ/cm2) provoked a reduction in Nrf2 protein level. A moderate decrease
in Keap1 protein level was also found (Chhabra et al. 2019). UVB exposure (20 mJ/cm2) of a
highly pigmented non-tumorigenic mouse melanoma cell line (Melan-a) resulted in reductions
of Keap1 and Nfr2 mRNA level after 6 h. Keap1 protein level was reduced as well. Both
markers were normalized within 24 h. In contrast, protein and mRNA of NQO1 were induced
at both time intervals, especially after 24 h from exposure (Kim et al. 2017). Nrf2 mRNA and
protein depletion was also found in human keratinocyte cell line HaCaT after treatment with
180 mJ/cm2 of UVB. Meanwhile, the level of HO-1 was up-regulated (Chen et al. 2019). In
contrast, UVB exposure of human keratinocyte (60 mJ/cm2) and fibroblast (200 mJ/cm2) cell
lines (CDD 1102 KERTr and CCD 1112Sk, respectively) increased the protein level of Nrf2,
HO-1 and reduced the level of Keap1 and Bach1 (Gęgotek et al. 2019; Gęgotek et al. 2016).
Although the effect of UVB on skin cells/tissue is ambiguous, studies on Nrf2-deficient mice
suggest an important role for Nrf2 in protecting against UVB radiation. Kawachi et al.
demonstrated that exposure of Nrf2 knockout mice to UVB (200 mJ/cm2) induced a stronger

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and longer lasting sunburn reaction as well as more pronounced histological changes,

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including epidermal necrosis, dermal oedema, inflammatory cell infiltration, sunburn and
apoptotic cell formation, and accumulation of 8-oxo-dGuo compared to Nrf2-wild-type mice

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(Kawachi et al. 2008). They also reported that UVB irradiation (200 mJ/cm2) accelerated
photoageing symptoms, such as coarse wrinkle formation, loss of skin flexibility, epidermal
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thickening, and the deposition of extracellular matrix in the upper dermis in Nrf2 knockout
mice. An increased level of the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) was
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found in the skin of the animals, as well as a decreased level of GSH (Hirota et al. 2011).
Other authors showed that, in Nrf2 knockout mice, UVB radiation (300 mJ/cm2) stimulated
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the overexpression of p53 protein, extracellular matrix degradation and short-term

inflammatory markers (IL-1 and IL-6) as well as the number of apoptotic cells (Saw et al.
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2011; Saw et al. 2014). The failure of UVB to activate Nrf2 may result from the fact that
UVB photons stimulate the direct formation of DNA lesions such as CPDs and 6-4PPs that
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strongly inhibit DNA transcription and, as a consequence, UVB probably delays transcription
of Nrf2-driven genes. Concurrently, increased levels of ROS and OS may contribute to the
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stimulation of apoptosis instead of the activation of survival mechanisms (Ikehata and

Yamamoto 2018).

Effect of UVA
Compared to UVB radiation, the uncompromising potential of UVA light to activate Nrf2
nuclear translocation and the expression of Nrf2-driven genes has been described in dermal
fibroblasts. UVA irradiation (25 J/cm2) of human dermal fibroblasts FEK4 stimulated Nrf2
increases in the cytosol and, mainly, in the nucleus. Expression of the Nrf2 target protein HO-
1 was enhanced as well (Zhong et al. 2010). However, repeated UVA treatment of fibroblasts
FEK4 reduced the transcriptional activation of HO-1 following irradiation. A phenomenon
known as refractoriness was accompanied by reduced accumulation of Nrf2 and increased
nuclear accumulation of Bach1, the Nrf2 repressor (Zhong et al. 2014). In mice skin explants,
a UVA1 dose of 40 J/cm2 increased nuclear Nrf2 accumulation and, in human neonatal skin
fibroblasts, also increased the expression of Nrf2, HO-1and GCL modifier subunit (GCLM)
genes. An analogous response was obtained by the treatment of cells with oxidised 1-
palmitoyl-2-arachidonyl-sn-glycerol-3-phosphorylcholine, the most abundant phospholipid in
the cell membrane, suggesting the involvement of lipid degradation products in UVA-caused
Nrf2 nuclear translocation (Gruber et al. 2010). Similarly, 4-HNE was shown to activate

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Nrf2-ARE signalling by changing Keap1 conformation (Siow et al. 2007). In UVA-irradiated

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(10 J/cm2) mouse fibroblasts, Nrf2 was clearly localized in the nucleus, while in non-

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irradiated cells the protein was in the perinuclear area in the cytosol. Translocation was
accompanied by an increased mRNA expression of Nrf2 target gene GCLM and its catalytic

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(GCLC) subunit (Hirota et al. 2005). Similarly, UVA exposure (5-7.5 J/cm2) significantly
stimulated Nrf2 nuclear translocation 3 h after treatment in human dermal fibroblasts FEK4.
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Further increases in HO-1 mRNA and protein levels, as well as NQO1 protein amounts, were
found in NHDF (Ryšavá et al. 2020). Up-regulation of Nrf2 and HO-1 was also detected in
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human fibroblasts NB1-RGB following exposure to 10 J/cm2 of UVA. However, the other
target protein, NQO1, was slightly reduced (Saito et al. 2015).
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In contrast to dermal fibroblasts, Nrf2 activation following UVA irradiation of epidermal

keratinocytes and melanocytes was not found in all studies. Treatment of NHEM, iMC23 and
iMC65 mouse melanocytes with UVA light (20 J/cm2) stimulated Nrf2 accumulation that was
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promoted by p62 up-regulation in the cells (Sample et al. 2018). UVA exposure (22.5 and
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45 J/cm2) of primary neonatal NHEK stimulated expression of the Nrf2-driven genes GCLC,
NQO1 and HO-1. In primary neonatal NHEM, an effect was observed on GCLM and HO-1.
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In both cell lines, combined treatment with UVA and UVB (480 and 960 mJ/cm2) was
obviously less efficient (Marrot et al. 2008). However, in UVA-treated (3 J/cm2) primary
neonatal NHEM, a moderate decrease in Nrf2 protein level was found. An obvious reduction
was also found in the level of Keap1 (Chhabra et al. 2019). UVA irradiation (8 J/cm2) of
B16F10 mouse melanoma cells caused a time-dependent reduction in nuclear Nrf2
translocation, but an increase in Nrf2 mRNA level; the peak was at 1 and 4 h, respectively.
This was accompanied by increased phosphorylation of p-ERK, p-JNK and p-p38, peaking
30 min after UVA irradiation. Specific kinase inhibitors significantly increased Nrf2
translocation. UVA also caused a significant decline in GCLC, GCLM, GST and NQO1
mRNA levels at 2 h after exposure and GCLC, GST and NQO1 protein levels and GST and
NQO1 activity at 6 h post-irradiation (Chaiprasongsuk et al. 2016). The same UVA dose also
reduced mRNA and protein expression of CAT and GPX after 2 and 6 h, respectively
(Onkoksoong et al. 2018). In normal primary mouse keratinocytes irradiated with UVA (5-20
J/cm2), activation of Nrf2 target genes or the Nrf2-dependent reporter gene was not observed
(Durchdewald et al. 2007). Similarly, UVA irradiation of mouse keratinocytes (20 J/cm2)
caused minimal up-regulation of the Nrf2-driven genes mRNA expression (HO-1, GCLC,
GCLM). A double UVA dose was more efficient (Zhao et al. 2013). Treatment of NHEK with
a UVA dose of 10-20 J/cm2 stimulated neither Nrf2 nuclear translocation nor its protein level.

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But surprisingly, mRNA and protein amounts of the target genes HO-1 and NQO1 were

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increased (Ryšavá et al. 2020). In human keratinocyte cell line (HaCaT), slightly increased

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Nrf2 protein expression and nuclear accumulation was demonstrated in cells exposed to 40
J/cm2 of UVA (Tian et al. 2011). Similarly, there were moderate increases in Nrf2, NQO1 and

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GCLC in HaCaT after UVA (15 J/cm2) exposure (Hseu et al. 2015a). On the other hand, a
recent study demonstrated a significant increase in Nrf2 and HO-1 protein and mRNA levels,
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which was accompanied by a Bach1 protein decrease in HaCaT cells irradiated with a similar
UVA dose of 16 J/cm2. NQO1 protein level was also enhanced, but only insignificantly (Yang
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et al. 2017b). Another study showed a rapid Nrf2 nuclear translocation within 1 h after UVA
irradiation, but reductions in HO-1 and NQO1 mRNA and protein levels (Ryšavá et al. 2020).
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An explanation for the different responses observed in epidermal cells is not clear. At least
partially, it may be related to the different genetic background of skin cells from different
donors. Mouse cells from various strains also have their own genetic characteristics, different
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from human ones. Another very important factor that plays a role in the cellular response to
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UVA exposure is the radiation source, as lamps from different manufacturers differ in
radiated wavelengths and their intensity. The UV spectrum is divided into components A, B
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and C rather formally. Therefore, the ability to penetrate the skin and biological effects of the
UVA2 region (320 - 340 nm) is closer to the UVB region than UVA1 (340 - 400 nm) (Diffey
et al. 2000, Edström et al. 2001), and thus the source generating UVA2 wavelengths will
stimulate different damage than sources emitting UVA1 or UVA. The dissimilar behaviour of
keratinocytes and fibroblasts is also probably associated with their genetic background and is
connected to their function in skin tissue. Keratinocytes that reside in the upper skin layer, the
epidermis, and are more exposed to environmental stressors display a higher antioxidant
capacity, important for the regulation of intracellular ROS level and other reactive
compounds. For example, (Applegate et al. 1995) and (Zhong et al. 2010) similarly
demonstrated a significantly higher level of constitutively expressed HO-2 enzyme in
keratinocytes compared to fibroblasts. Further, the level of the most important non-enzymatic
antioxidant GSH that is under the control of Nrf2 transcription factor, was about 30 % higher
in keratinocytes than in fibroblasts (Niggli and Applegate 1997; Tyrrell and Pidoux 1988). A
study by Niggli and Applegate also showed a minimal decrease in GSH in keratinocytes
(90 % of control) compared to fibroblasts (57 % of control) following exposure to a UVA
dose of 40 J/cm2 (Niggli and Applegate 1997). As a result, different Nrf2 activation/activity
can be found in individual cells, which manifests in their diverse sensitivity and response to
UVA radiation (Fig. 5).

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Pharmacological Activation of Nrf2

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Pharmacological activation of Nrf2 transcription factor seems to be an efficient strategy for
suppressing OS associated with massive ROS generation in various tissues and organs,

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including skin. Currently, more than 200 compounds have been reported to activate the Nrf2
protein in diverse cell types and stimulate its target genes expression (Victor et al. 2020). The
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activators include synthetic compounds as well as natural molecules commonly occurring in
plants or food. To date, the most potent known Nrf2 activators are the semi-synthetic
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cyanoenone triterpenoids, designed from the natural product oleanolic acid (Dayalan Naidu
and Dinkova-Kostova 2020). The best described natural Nrf2 activator is sulforaphane
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(Briones-Herrera et al. 2018; Cuadrado et al. 2019). Nrf2 activator dimethyl fumarate is
marketed as a treatment for melanoma, the skin disorder psoriasis and some chronic
inflammatory diseases, with the others under pre-or clinical trials for treatment of the same
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(Cuadrado et al. 2019).

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Natural Nrf2 Activators in UVA and UVB Photoprotection

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As mentioned above, skin covers our body and protects us against assaults from
environmental factors. Solar radiation acts continuously every day and represents the main
factor that contributes to ROS/RNS production. Powerful elimination of oxidative compounds
enhances skin tissue regeneration and can prevent skin ageing (including modification of both
cellular and non-cellular elements), malignant cell transformation and cancer development
(Fig. 4). Therefore, molecules that activate Nrf2 transcription factor seems to be useful in this
area of skin care.
 Currently, the use of pure natural compounds or extracts, as opposed to chemically
synthesized substances, is becoming very popular in dermatological preparations, including
those for photoprotection (Yamamoto et al. 2018). The main accepted mechanism of the
compounds’ effects on the Nrf2 signalling pathway is associated with their antioxidant
properties and can be divided into two categories. One category includes electrophilic indirect
Nrf2 activators that covalently bind to the thiol of the cysteine residue(s) in the dimerization
domain of Keap1. The binding of Nrf2 activator to Keap1 causes conformational changes in
Keap1, which trigger the dissociation of the Keap1-Nrf2 complex, Nrf2 release and its
translocation to the nucleus. The second category comprises non-electrophilic direct Nrf2
activators that bind directly to the Nrf2 binding domain of Keap1 (thus disrupting the Keap1-

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Nrf2 complex) and also stimulate Nrf2 nuclear translocation (Chu et al. 2017). Due to the

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pleotropic properties of natural compounds, they can modulate different control proteins of

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the Nrf2 signalling pathway such as FynK, Cav-1, Bach1 and others and thus influence the
nuclear import/export of Nrf2 and its control proteins. Natural compounds can also provoke

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activation of protein kinases such as PKB, PKC, MAPK, AMPK or PERK that cause Nrf2
phosphorylation, Keap1-Nrf2 complex disintegration and Nrf2 nuclear accumulation
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(Gęgotek and Skrzydlewska 2015; Huang et al. 2002; Chun et al. 2014; Yang et al. 2021).
However, most published studies on cyto- and/or photoprotection do not include natural
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compounds effects on these proteins, they instead mainly investigate their effects on Nrf2-
driven genes protein activity/level and/or mRNA expression.
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A brief up-to-date summary of the identified natural Nrf2 activators beneficial for skin cell
(fibroblasts and keratinocytes) photoprotection is presented in Tables 1-4. Some of the
compounds may be classified as endogenous molecules as they are produced in the human
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body as well, for example, α-melanocyte-stimulating hormone (α-MSH) (Kokot et al 2009),

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melatonin and its derivatives (Slominski et al. 2004; Skobowiat et al. 2018) and vitamin D
and its derivatives (Chaiprasongsuk et al. 2019; Chaiprasongsuk et al. 2020; Slominski et al.
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2020b). Concerning melanocytes, photoprotection is only marginally evaluated. Most studies

in relation to Nrf2 translocation and activation of driven genes unfortunately use hydrogen
peroxide to provoke oxidative stress. However, a few papers showed protective effects against
UVA/UVB radiation such as for caffeic acid, ferulic acid, quercetin and rutin
(Chaiprasongsuk et al. 2016), melatonin or its metabolites (Janjetovic et al. 2017) and
nicotinamide (Chhabra et al. 2019).
Unfortunately, a majority of studies (Tables 1-4) tested the effects of compounds and/or
extracts on the Nrf2 signalling pathway with only the use of simple in vitro or possibly ex vivo
systems; in vivo experiments only appear in the literature sporadically, such as trials
concerning the photoprotective effects of SFN and hispidulin (Chaiprasongsuk et al. 2017b),
and cannabidiol (Atalay et al. 2021). As such, animal and clinical trials especially will be
necessary to validate the effectiveness and suitability of new compounds as candidates for
photoprotective applications. Such studies are also essential to exclude any possible harmful
effects on skin or other tissues.

Conclusion
In summary, the available studies suggest that UVB radiation is a rather poor activator or
even inhibitor of the Nrf2 signalling pathway in epidermal keratinocytes and melanocytes, as

t
well as dermal fibroblasts. In contrast, UVA radiation stimulates Nrf2 activation in dermal

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fibroblasts. Nevertheless, in keratinocytes and melanocytes, the effect of UVA on the Nrf2

cr
signalling pathway remains unclear. UVA photons, with a longer wavelength, are more
penetrating and thus can reach deeply localized fibroblasts, provoking oxidative damage to

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them. From this point of view, Nrf2 activation in fibroblasts is of greater importance in
comparison to keratinocytes and melanocytes, which are mainly exposed to the action of
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UVB photons, with its particularly harmful effects on DNA. Studies concerning Nrf2 further
suggest that constitutive or long-term Nrf2 activation in keratinocytes is detrimental due to
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abnormal enhancement of keratinocyte differentiation. Therefore, exogenous modulation of

the Nrf2 signalling pathway in the skin tissue by (phyto)chemicals must be performed
ed

carefully in order to achieve beneficial effects while preventing possible harmful effects on
cells (as Nrf2 activation involves many control/target proteins). Primarily, the health status of
the skin (physiological and pathological) and long-term/chronic application of dermal
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preparations containing Nrf2 modulators must be considered. Thus, the safe dermal use of
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Nrf2 modulators requires a wide knowledge of their molecular targets, not only their effects
on Nrf2-ARE-controlled genes.
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Acknowledgments
This work was financially supported by grant IGA_LF_2020_022, IGA_LF_2021_011 and
the Institutional Support of Palacký University in Olomouc - RVO 61989592.

Conflict of Interest Statement:

The authors state that there are no conflicts of interest regarding the publication of this article.
Notes on contributors
Alena Ryšavá is a Ph.D. student at Department of Medical Chemistry and Biochemistry,
Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic.
Jitka Vostálová, Ph.D., is a researcher and associate professor at Department of Medical
Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University,
Olomouc, Czech Republic.
Alena Rajnochová Svobodová, Ph.D., is a researcher and associate professor at Department of
Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University,
Olomouc, Czech Republic.

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Abbreviations
4-HNE 4-Hydroxynonenal
6-4 PP Pyrimidone photoproduct
ABC ATP-binding cassette (transporter)
Akt Serine/threonine kinase
AMPK AMP-activated protein kinase
AP-1 Activator protein-1
ARE Antioxidant response element
Arg Arginine
Asp Aspartic acid

t
Bach1 Transcription factor BTB and CNC homology 1

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Bach2 Transcription factor BTB and CNC homology 2

cr
BRG1 Brahma-related gene 1
bZIP Basic leucine zipper
CAT
Cav-1
Catalase
Caveolin-1 us
an
CBP Binding protein
CBR1 Carbonyl reductase 1
M

CHD6 Chromo-ATPase/helicase DNA binding protein

CK2 Casein kinase 2
Cap’n’Collar
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CNC
CPD Cyclobutane pyrimidine dimer
CREB cAMP response element binding protein
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Crm1 Chromosomal region maintenance-1 protein

ce

Cul3 Cullin3-based E3 ligase

Cys Cysteine
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DDB2 Damaged-DNA binding protein 2

EpRE Electrophile response element
ERK Extracellular signal-regulated kinase
EPHX1 Epoxide hydrolase 1
Fos Proto-oncogenic transcription factor
FynK Fyn kinase
GCS Glutamylcysteine synthetase
GCLC Glutamate-cysteine ligase catalytic subunit
GCLM Glutamate-cysteine ligase modifier subunit
Gln Glutamine
Glu Glutamic acid
GPX Glutathione peroxidase
GSH Reduced glutathione
GSK-3 Glycogen synthase kinase-3
GSR Glutathione reductase
GSSG Oxidized glutathione
GST Glutathione S-transferase
GSTPi Glutathione S-transferase pi

t
HaCaT Human skin keratinocyte cell line

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HO-1 Heme oxygenase 1

cr
HO-2 Heme oxygenase 2
IL Interleukin
JNK
Jun
c-Jun N-terminal kinase
Proto-oncogenic transcription factor us
an
K Keratin
Keap1 Kelch-like ECH-associated protein 1
M

KGF Keratinocyte growth factor

KIR Keap1-interacting region
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LC Langerhans cell
Leu Leucine
MAPK Mitogen-activated protein kinase
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Melan-a Non-tumorigenic mouse melanoma cell line

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MMP Matrix metalloproteinase

α-MSH alfa-Melanocyte stimulating hormone
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mTORC1 Rapamycin complex 1

NAD(P)H Nicotinamide adenine dinucleotide (phosphate) reduced
Neh Nrf2-ECH homology domain
NFκB Nuclear factor kappa B
NHDF Normal human dermal fibroblast
NHEK Normal human epidermal keratinocyte
NHEM Normal human epidermal melanocyte
NQO1 NAD(P)H quinone oxido-reductase 1
Nrf1 Nuclear factor-erythroid 2 (NF-E2)-related factor 1
Nrf2 Nuclear factor-erythroid 2 (NF-E2)-related factor 2
Nrf3 Nuclear factor-erythroid 2 (NF-E2)-related factor 3
OS Oxidative stress
8-oxo-dGuo 8-Oxo-7,8-dihydro-2´-deoxyguanosine
P Phosphorylation
p45 NF-E2 p45 Nuclear factor erythroid-derived 2
PERK PKR-like endoplasmic reticulum kinase
PKB Protein kinase B
PKC Protein kinase C

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PKI3 Phosphoprotein kinase 3

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pNrf2 Phosphorylated (Ser40) Nrf2

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Pro Proline
PRX Peroxiredoxin
Rbx1
RNS
Glutathione S-transferase-tagged Roc1 protein
Reactive nitrogen species us
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ROS Reactive oxygen species
RXR-α Retinoid X receptor alfa
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Ser Serine
sMaf Small musculo-aponeurotic fibrosarcoma protein
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SOD Superoxide dismutase

TAK1 Transforming growth factor-β-activated kinase 1
TFEB Transcription factor EB
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Thr Threonine
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TNF-α Tumor necrosis factor-alfa

β-TrCP beta-Transducing repeat-containing protein
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TRX Thioredoxin
TrxR Thioredoxin reductase
Tyr Tyrosine
U Ubiquitin
UGT UDP-glucuronosyltransferase
UV Ultraviolet radiation
Figure legends

Figure 1. Schematic structure of human skin.

Human skin is comprised of epidermis, dermis and hypodermis. The epidermis contains
keratinocytes, melanocytes and Langerhans cells that are critical for the structural and
functional integrity of the epidermis. In the epidermis, five layers of keratinocytes can be
distinguished: stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and
stratum basale. Each layer is characterized by the expression of specific proteins, including
(cyto)keratins (K), neutral-basic K1-K6 and acidic K10-K17. The strata granulosum,
spinosum and basale contain living keratinocytes, whereas the strata corneum and lucidum

t
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are formed only from dead keratinized cells. Melanocytes reside in the stratum basale and are
responsible for synthesis of the pigment melanin and its transport to surrounding

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keratinocytes in the form of particles called melanosomes. Langerhans cells represent skin

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immunity elements and are located mostly in the stratum spinosum. The basal membrane
separates the epidermis from the dermis, where there are fibroblasts that synthesize several
structural proteins including collagen and elastin. The deepest layer, the hypodermis, consists
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of loose connective tissue, elastin and collagen fibres, fibroblasts, adipocytes and
macrophages. The hypodermis and dermis are vascularized and contain nerve endings, sweat
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and sebaceous glands, hair follicles and lymphatic vessels.

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Figure 2. Schematic structure of Nrf2 protein and its post-translational modification by

kinases.
pt

Nrf2 contains seven Nrf2-ECH homology (Neh) domains with different functions. The Neh2
domain is located at the N-terminus and interacts with Kelch-like ECH-associated protein 1
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(Keap1) through the DLG and ETGE motifs. Seven lysin residues (K) are important for
ubiquitylation. The Neh4 and Neh5 domains are transactivation domains for cAMP response
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element binding protein (CREB), binding protein (CBP) and Brahma-related gene 1 (BRG1).
The Neh7 domain is involved in the binding of retinoid X receptor α (RXR-α). The Neh6
domain interacts with β-transducing repeat-containing protein (β-TrCP) through the DSGIS
and DSAPGS motifs. The Neh1 is a dimerization domain with small musculo-aponeurotic
fibrosarcoma proteins (sMaf) and CNC-bZIP domain with DNA binding to antioxidant
responsive element (ARE). The Neh3 domain is located at the C-terminus and represents a
transactivation domain with chromo-ATPase/helicase DNA binding protein (CHD6).
Nrf2 activity is regulated by several kinases. Phosphorylation of Nrf2 by protein kinase C
(PKC) at Ser40 residue, PKR-like ER kinase (PERK) at Thr80 residue, casein kinase 2 (CK2)
at multiple sites and AMP-activated protein kinase (AMPK) at Ser558 residue increase Nrf2
nuclear translocation and related gene activation. On the other hand, glycogen synthase kinase
(GSK-3β) phosphorylates Nrf2 at Ser344 and Ser347 residues that stimulates Nrf2
ubiquitination and degradation. Fyn kinase (FynK), after activation by GSK-3β,
phosphorylates Nrf2 present in the nucleus at Tyr576 residue and provokes its export via
exportin 1 to the cytosol and its proteasomal degradation. Mitogen-activated protein kinases
(MAPKs), particularly C-Jun N-terminal kinase (JNK), extracellular signal regulated kinase
(ERK) and p38 phosphorylate Nrf2 at Ser215, Ser408, Ser558 and Ser577 or Thr 559 residues

t
and positively or negativity modulate Nrf2 activity.

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Figure 3. Schematic diagram of Nrf2 activity regulation.

Under basal (non-stress) conditions, Nrf2 is kept in a complex with Keap-1 that mediates Nrf2

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ubiquitination and proteasomal degradation. Keap-1 is released (Keap1-pool) and can bind
newly synthetized Nrf2. Degradation of Nrf2 is also stimulated by binding of -TrCP to the
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Neh6 domain of Nrf2 and subsequent phosphorylation via GSK-3. Under OS conditions, the
Nrf2-Keap1 complex is modified due to the interaction of ROS and/or electrophiles with
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specific SH-groups on Keap1 (Keap1 ox). This results in either disruption of Nrf2-Keap1
interaction or inability of Keap1 regeneration to bind Nrf2 and thus Nrf2 may translocate to
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the nucleus. Nrf2 in complex with Keap1 may also be phosphorylated (Nrf2-P) by protein
kinases (e.g., PKC at Ser40, PERK at Thr80, AMPK at Ser558, CK2 at multiple sites) that
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stimulates Nrf2 release. ROS also stimulate decreases in the membrane protein caveolin 1
(Cav-1) level and its phosphorylation (Cav-1-P), consequently increasing free Nrf2 in the
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cytosol and facilitating Nrf2 nuclear accumulation. Binding of p62 to Keap-1 also stimulates
destruction of Nrf2-Keap1 interactions and Nrf2 release. Protein p21 is able to interact with
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Nrf2, prevents its ubiquitination and increases its stabilization. Released Nrf2 and/or Nrf2-P
translocates to the nucleus and competes with the transcription regulatory protein Bach1,
which normally represses Nrf2-driven gene transcription, for binding to the promoter
antioxidant response element (ARE) site. As a result of increased Nrf2 nuclear levels, the
interaction of Bach1 with small musculo-aponeurotic fibrosarcoma Maf proteins (sMaf) is
destroyed and Bach1 is released. Nrf2 interacts with sMaf and the resulting complex
stimulates transcription of ARE-controlled genes, which have antioxidant and/or
cytoprotective effects. In addition, Nrf2 stimulates its own expression as well as of Keap-1.
Released Bach1 is phosphorylated by extracellular signal-regulated kinases 1/2 (ERK1/2) at
Tyr486 or possibly by other kinases that stimulates Bach1 nuclear export through exportin-1
and a resulting degradation in the proteasome. As a delayed response to OS, glycogen
synthase kinase-3β (GSK-3) is activated by phosphorylation at Tyr216 via the
phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) cascade in the cytosol. The
phosphorylated GSK-3 (GSK-3-PTyr216) acts as an upstream regulator of Src-family
kinases, including Fyn kinase (FynK), and participates in the renewal of redox homeostasis.
GSK-3-PTyr216 phosphorylates FynK at Thr residue(s). The phosphorylated FynK (FynK-
PThr) translocates to the nucleus and phosphorylates Nrf2 at Tyr576 resulting in Nrf2-PTyr576

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release from ARE, Nrf2-PTyr576 nuclear export and its proteasomal degradation. Thus FynK-

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PThr decreases Nrf2-driven gene transcription. FynK-PThr is then phosphorylated by itself or an

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unknown kinase at Tyr213 (FynK PThr-PTyr213), which stimulates its nuclear export and
degradation in the proteasome.

Figure 4. Nrf2 and Keap1 cooperation in human epidermis. us

an
While Nrf2 level is uniform from the stratum basale to stratum granulosum, the amount of
Keap1 is highest in the stratum basale and decreases towards the stratum granulosum. Due to
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this, less Nrf2 is bound in the complex with Keap1 in the stratum granulosum compared to
the stratum basale. Consequently, Nrf2 activity and thus function is different in individual
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epidermal layers, resulting in a preference for apoptosis activation in damaged basal

keratinocytes and cytoprotective gene transcription in damaged granular keratinocytes.
pt

Figure 5. Effect of UVA radiation on Nrf2 localization in NHDF and NHEK.

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Data obtained using of primary cells from two donors are presented to demonstrate different
response to UVA (315–380 nm) irradiation. Nrf2 cellular localization was determined 3 h
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after exposure. Immunocytochemical staining of Nrf2 protein (brown) in normal dermal

human fibroblasts (NHDF) exposed to a UVA dose of 7.5 J/cm2 (100-fold magnification) and
normal human epidermal keratinocytes (NHEK) exposed to 10 J/cm2 (200-fold magnification)
is shown. Haematoxylin was used for nuclei staining (blue). Data are partially adapted from
our recent report (Ryšavá et al. 2020).
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Table 1. Phytochemicals as Nrf2 modulators in UVA irradiated human keratinocytes.
UVA dose
Compound Concentration Effect Ref.
[J/cm2]
HaCaT
Acetyl-11-keto-β- ↑ Nrf2, HO-1, NQO1, GST (Yang et al.
2.5 µM 16
boswellic acid ↓ Bach1 2017b)
Bromophenethyl ↑ Nrf2, HO-1 (Kuo et al.
50 µM 10
caffeamide ↓ MMP1 2020)
↑ GCL (Pluemsamran
Caffeic acid 15 µM 4
↓ MMP1 et al. 2012)
↑ Nrf2, HO-1, NQO1, GCL (Hseu et al.
Ectoine 1.5 µM 3
↓ Keap1 2020)
(Hseu et al.
Ellagic acid 50 µM 20 ↑ Nrf2, HO-1, Keap1, SOD 2012; Yang et
al. 2021)

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↑ Nrf2, HO-1, NQO1, GCL (Hseu et al.

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Ergothioneine 500 nM 15
↓ Keap1 2015a)
↑ CAT (Pluemsamran

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Ferulic acid 30 µM 4
↓ MMP1, GCL et al. 2012)
Hexaguluroic acid (Li et al.
10 µM 30 ↑ Nrf2

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hexasodium salt 2020a)
↑ Nrf2, HO-1, NQO1, GCL (Hseu et al.
Lucidone 4 µM 15
↓ Keap1 2015b)
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Phenethyl ↑ Nrf2, HO-1, (Chu et al.
50 µM 10
caffeamide ↓ MMP1, MMP2 2019)
↑ Nrf2 (Deng et al.
Pterostilbene 9.75 µM 20
↓ HO-1, CAT
M

2018)
(Kimura et al.
2009;
Quercetin 50 µM 30 ↑ Nrf2 Rajnochová
ed

Svobodová et
al. 2017)
↑ Nrf2 (Liu et al.
Resveratrol 10.95 µM 2.8
↓ Keap1 2011)
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Tetramannuronic
(Li et al.
acid tetrasodium 10 µM 30 ↑ Nrf2
2020b)
ce

salt
↑ Nrf2, HO-1 (Yang et al.
Zerumbone 10 µM 15
↓ Keap1 2018)
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CDD1102KERTr
↑ pNrf2, HO-1, NFκB (Jastrząb et al.
Cannabidiol 1 µM 30
↓ Keap1, Bach1 2019)
CDD1102KERTr with HPV-16 E6/E7
↑ pNrf2, HO-1, Keap1 (Gęgotek et
Ascorbic acid 100 µM 30
↓ Bach1 al. 2019)
↑ pNrf2, HO-1 (Gęgotek et
Rutin 25 µM 30
↓ Keap1, Bach1 al. 2019)
Sea buckthorn (Gęgotek et
500 ng/ml 30 ↑ pNrf2, HO-1
seed oil al. 2018)
Table 2. Phytochemicals as Nrf2 modulators in UVA irradiated human fibroblasts.

UVA dose
Compound Concentration Effect Ref.
[J/cm2]
NHDF
↑ MMP1 (Hahn et al.
Ferulic Acid 10 µM 10
↓ CAT, SOD 2016)
(Chaiprasongsuk
↑ Nrf2, NQO1, GCL, GST et al. 2017a;
Hesperetin 15 µM 8
↓ Nrf2, MMP1 Lohakul et al.
2021)

Paeoniflorin 800 µM 22.5 ↑ Nrf2, HO-1, NQO1 (Lu et al. 2020)

Pyrroloquinoline ↑ Nrf2 (Zhang et al.

0.15 µM 9
quinine ↓ HO-1 2015)
(Svobodová et al.

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Silybin 12.5 µM 7.5 ↑ HO-1, MMP1
2018)

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Tricaffeoylaltraric (Parzonko and
50 µM 25 ↑ Nrf2, HO-1
acid Kiss 2019)

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Tremella fuciformis ↑ Nrf2, HO-1, NQO1, SOD, CAT
12.5 mg/ml 12 (Fu et al. 2021)
polysaccharides ↓ Keap1

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Harak herbal ↑ NQO1, GST (Lohakul et al.
30 µg/ml 8
formula ↓ Nrf2 2021)
CCD1112Sk
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↑ Nrf2, pNrf2, HO-1, NFκB (Gęgotek et al.
Ascorbic acid 100 µM 20
↓ Keap1, Bach1 2019 and 2017)
↑ Bach1 (Gęgotek et al.
Rutin 25 µM 20
↓ pNrf2, HO-1, Keap1
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2019)
Sea buckthorn seed (Gęgotek et al.
500 ng/ml 20 ↑ pNrf2, HO-1
oil 2018)
GT-F
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(Offord et al.
↑ HO-1
Carnosic acid 0.3-1 µM 50 2002; Park et al.
↓ MMP1 2013)
(Offord et al.
pt

Lycopene 0.5-1 µM 50 ↑ HO-1, MMP1

2002)
CCD986Sk
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Porphyra-334 10 µM 10 ↑ Nrf2, HO-1, GST (Ryu et al. 2015)

HFP-1
(Obermüller-
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Jevic et al. 2001

β-Carotene 0.5-1 µM 20 ↑ HO-1, MMP1
and 1999; Offord
et al. 2002)
HSF
↑ Nrf2
Zerumbone 8 µM 3 (Hseu et al. 2019)
↓ Keap1, MMP1
Hs68
↑ Nrf2, HO-1
t-Cinnamic acid 100 µM 3 (Hseu et al. 2018)
↓ MMP1
NB1-RGB
↑ HO-1
Green propolis 30 µg/ml 10 (Saito et al. 2015)
↓NQO1
Table 3. Phytochemicals as Nrf2 modulators in UVB irradiated human keratinocytes.

UVB dose
Compound Concentration Effect Ref.
[mJ/cm2]
HaCaT
3,5-Dicaffeoyl-epi- ↑ Nrf2
10 µM 15 (Oh et al. 2019)
quinic acid ↓ SOD, HO-1
(Chen et al.
6-Shogaol 20 µM 180 ↑ Nrf2, HO-1 2019)
↑ Nrf2, HO-1, NQO1 (Wang et al.
Baicalein 10 µM 125
↓ NFκB 2018)
Epigallocatechin-3- (Park et al.
4 µM 15 ↑ Nrf2, HO-1, SOD1, NFκB 2021)
gallate
(Hewage et al.
↓ Nrf2, GCLC

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Gelangin 40 µM 30 2017)

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Ginsenoside Rg1 50 µM 60 ↑ Nrf2, HO-1, GCL, NFκB (Li et al. 2016)

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(Park et al.
Malonic acid 4 µM 15 ↑ Nrf2, HO-1, SOD1, NFκB 2021)

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(Fernando et al.
Rosmarinic acid 2.5 µM 30 ↓ Nrf2, SOD, CAT, HO-1 2016)
↑ MMP1 (Kim et al.
Sulforaphane 10 µM 15
↓ Nrf2 2015)
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↑ Nrf2, HO-1, NQO1, MMP1 (Wang et al.
Wogonin 10 µM 125
↓ NFκB 2018)
↑ Nrf2, HO-1, SOD1, MMP1, MPP2 (Kim et al.
M

Youngiaside A, C 10 µM 15 2015)

Elderberry extract 10 µg/ml 125 ↑ Nrf2, HO-1, NQO1, MMP1, NFκB (Lin et al. 2019)
ed

(Fernando et al.
Fucoidan 50 µg/ml 50 ↑ Nrf2, HO-1 2020)
Red raspberry (Wang et al.
200 µg/ml 100 ↑ Nrf2, HO-1, SOD, CAT 2019)
extract
pt

Scutellaria
(Wang et al.
baicalensis Georgi 10 µg/ml 125 ↑ Nrf2, HO-1, NQO1, MMP1, NFκB 2018)
ce

radix extract
(Park et al.
Walnut seed extract 100 µg/ml 50 ↓ Nrf2, HO-1, NQO1 2014)
Youngia
↑ Nrf2, HO-1, SOD1, MMP1, (Kim et al.
Ac

denticulatum 10 µg/ml 15 2015)

MMP2
extract
CDD1102KERTr
↑ pNrf2, HO-1, NFκB (Jastrząb et al.
Cannabidiol 1 µM 60
↓ Keap1, Bach1 2019)
CDD1102KERTr with HPV-16 E6/E7
↑ pNrf2, HO-1, Keap1, NFκB (Gęgotek et al.
Ascorbic acid 100 µM 60
↓ Bach1 2019)
↑ pNrf2, HO-1, NFκB (Gęgotek et al.
Rutin 25 µM 60
↓ Keap1, Bach1 2019)
Sea buckthorn seed ↑ pNrf2, HO-1 (Gęgotek et al.
500 ng/ml 60
oil ↓ Keap1 2018)

Table 4. Phytochemicals as Nrf2 modulators in UVB irradiated human fibroblasts.

UVB dose
Compound Concentration Effect Ref.
[mJ/cm2]
NHDF
↑ Nrf2, HO-1, NQO1 (Gao et al.
Acteoside 10 µM 144
↓ MMP1 2018c)

(Huang et al.
Baicalein 10 µM 15 ↓ Nrf2, HO-1, MMP1 2019)
↑ Nrf2, HO-1, NQO1 (Liu et al.
Ginsenoside C-Y 20 µM 144
↓ MMP1 2019b)
↑ Nrf2, HO-1, NQO1 (Gao et al.

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Quercetin 10 µM 144
↓ MMP1 2018a)

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↑ Nrf2, MMP1 (Huang et al.
Sulphoraphane 1 µM 15
↓ HO-1 2019)

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↑ Nrf2, HO-1, NQO1 (Liu et al.
Vina-ginsenoside R7 20 µM 144
↓ MMP 2019a)
Alchemilla mollis
extract
Borago officinalis L.
100 µg/ml

10 µg/ml
144

144
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↑ Nrf2, HO-1, NQO1
↓ MMP1

↑ Nrf2, HO-1, NQO1, MMP1

(Hwang et al.
2018)

(Seo et al.
an
extract 2018)
Foeniculum vulgare (Sun et al.
10 µg/ml 144 ↑ Nrf2, MMP1 2016)
Mill extract
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Helianthus annuus L. (Hwang et al.

10 µg/ml 144 ↑ Nrf2, HO-1, NQO1, MMP1 2019)
extract
Orobanche cernua (Gao et al.
10 µg/ml 144 ↑ Nrf2, HO-1, NQO1, MMP1
ed

loefling extract 2018c)

Prunus yeonesis ↑ HO-1, NQO1 (Li et al.
10 µg/ml 144
blossom extract ↓ Nrf2, MMP1 2018a)
pt

Pterocarpus ↑ Nrf2, HO-1, NQO1 (Gao et al.

10 µg/ml 144
santalinus L. extract ↓ MMP1 2018a)
Pueraria montana ↑ Nrf2, Keap1 (Heo et al.
ce

50 µg/ml 20
extract ↓ NQO1 2019)
Ribes nigrum L. ↑ HO-1 (Li et al.
10 µg/ml 144
extract ↓ Nrf2, IL-6, MMP1 2018b)
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Rubus idaeus L. ↑ Nrf2, HO-1, NQO1 (Gao et al.

10 µg/ml 144
Extract ↓ MMP1, NFκB 2018b)
CCD986Sk
↑ Nrf2 (Shin et al.
Geniposide 30 µM 70
↓ MMP2 2018)
CCD1112Sk
(Gęgotek et al.
↑ Nrf2, pNrf2, HO-1, NFκB
Ascorbic acid 100 µM 200 2019 and
↓ Keap1, Bach1 2017)
↑ pNrf2, HO-1, NFκB (Gęgotek et al.
Rutin 25 µM 200
↓ Keap1, Bach1 2019)
 ↑ pNrf2, HO-1 (Gęgotek et al.
Sea buckthorn seed oil 500 ng/ml 200
↓ Keap1 2018)
HS68
Jasminum sambac (Ho et al.
2.5 % 40 ↑ Nrf2, pNrf2, HO-1 2021)
flowers extract

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