A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism.

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Photooxidation of Tryptophan and Tyrosine Residues in Human Serum Albumin Sensitized by Pterin: A Model for Globular Protein Photodamage in Skin.

Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin.

Importance of UDP-glucuronosyltransferase 1A1 expression in skin and its induction by UVB in neonatal hyperbilirubinemia.

UDP-glucuronosyltransferase (UGT) 1A1 is the sole enzyme that can metabolize bilirubin. Human infants physiologically develop hyperbilirubinemia as the result of inadequate expression of UGT1A1 in the liver. Although phototherapy using blue light is effective in preventing jaundice, sunlight has also been suggested, but without conclusive evidence, to reduce serum bilirubin levels. We investigated the mRNA expression pattern of human UGT1A1 in human skin, human skin keratinocyte (HaCaT) cells, and skin of humanized UGT1 mice. The effects of UVB irradiation on the expression of UGT1A1 in the HaCaT cells were also examined. Multiple UGT1A isoforms, including UGT1A1, were expressed in human skin and HaCaT cells. When HaCaT cells were treated with UVB-exposed tryptophan, UGT1A1 mRNA and activity were significantly induced. Treatment of the HaCaT cells with 6-formylindolo[3,2-b]carbazole, which is one of the tryptophan derivatives formed by UVB, resulted in an induction of UGT1A1 mRNA and activity. In neonates, the expression of UGT1A1 was greater in the skin; in adults, UGT1A1 was expressed mainly in the liver. Treatment of humanized UGT1 mice with UVB resulted in a reduction of serum bilirubin levels, along with increased UGT1A1 expression and activity in the skin. Our data revealed a protective role of UGT1A1 expressed in the skin against neonatal hyperbilirubinemia. Sunlight, a natural and free source of light, makes it possible to treat neonatal jaundice while allowing mothers to breast-feed neonates.

Induction of long interspersed nucleotide element-1 (L1) retrotransposition by 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct.

Long interspersed nucleotide element-1 (L1) is a retroelement comprising about 17% of the human genome, of which 80-100 copies are competent as mobile elements (retrotransposition: L1-RTP). Although the genetic structures modified during L1-RTP have been clarified, little is known about the cellular signaling cascades involved. Herein we found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of the aryl hydrocarbon receptor (AhR), induces L1-RTP. Notably, RNA-interference experiments combined with back-transfection of siRNA-resistant cDNAs revealed that the induction of L1-RTP by FICZ is dependent on AhR nuclear translocator-1 (ARNT1), a binding partner of AhR, and the activation of cAMP-responsive element-binding protein. However, our extensive analyses suggested that AhR is not required for L1-RTP. FICZ stimulated the interaction of the L1-encoded open reading frame-1 (ORF1) and ARNT1, and recruited ORF1 to chromatin in a manner dependent on the activation of mitogen-activated protein kinase. Along with our additional observations that the cellular cascades for FICZ-induced L1-RTP were different from those of L1-RTP triggered by DNA damage, we propose that the presence of the cellular machinery of ARNT1 mediates L1-RTP. A possible role of ARNT1-mediated L1-RTP in the adaptation of living organisms to environmental changes is discussed.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy.

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues.

Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after approximately 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR *-. Additional mechanisms that may lead to the near disappearance of Trp(*) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.

Mechanism of the very efficient quenching of tryptophan fluorescence in human gamma D- and gamma S-crystallins: the gamma-crystallin fold may have evolved to protect tryptophan residues from ultraviolet photodamage.

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated beta-sheet Greek key domains of beta- and gamma-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human gammaD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552-11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human gammaS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n - 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.

Light-induced oxidation of tryptophan and histidine. Reactivity of aromatic N-heterocycles toward triplet-excited flavins.

Mechanisms of flavin-mediated photooxidation of electron-rich amino acids tryptophan and histidine were investigated for aqueous solutions. Indole, representing the tryptophan side chain in proteins, reacted at nearly diffusion controlled rates (k approximately 2.7 x 10(9) L mol(-1) s(-1) at 293 K) with the triplet-excited flavin state, but reactions of imidazole (and histidine) were significantly slower (k < 2.0 x 10(8) L mol(-1) s(-1)) as determined by laser flash photolysis. Oxidation rates of derivates were invariably susceptible to electronic factors affecting incipient radical cation stability, while no primary kinetic hydrogen/deuterium isotope effect was observed for imidazole. Thus reaction by electron transfer was proposed in contrast to a direct hydrogen abstraction. Unlike indole compounds, imidazole derivatives suffered from the presence of a basic imino nitrogen (=N-), which caused the rate constant of histidine free base (k approximately 1.8 x 10(8) L mol(-1) s(-1)) to drop considerably upon protonation. Complexation of the imino nitrogen with transition metals provoked changes in reactivity, as rate constants decreased after addition of Zn(2+) (k of 4-methylimidazole, as histidine model, decreased from 9.0 x 10(8) L mol(-1) s(-1) in the absence to 4.1 x 10(8) L mol(-1) s(-1) in the presence of ZnCl(2)). The pyrrole nitrogen (-NH-) was not directly involved in complexation reactions, but its electron density increased upon interaction with hydrogen bond-accepting anions and resulted in higher rate constants (k of 4-methylimidazole increased from 9.0 x 10(8) L mol(-1) s(-1) to 2.0 x 10(9) L mol(-1) s(-1) after addition of NaOAc). The high rate constants were in agreement with a large thermodynamical driving force, as calculated from oxidation peak potentials determined electrochemically. After oxidation, resulting radical cations were readily deprotonated and trapped by 2-methyl-2-nitrosopropane, as detected by electron paramagnetic resonance spectroscopy. Indole-derived spin adducts were attributed to selective trapping of C(3)-centered radicals, whereas spin adducts with imidazole-derivatives arose from both carbon and nitrogen-centered imidazolyl radicals.

Eye lens color: formation and function.

Aromatic amino acids are photooxidized by near-ultraviolet light to colored products that are bound very tightly to protein amino groups. The resulting colored proteins absorb near-ultraviolet light more strongly and are rendered more hydrophobic than the untreated compounds, and they fluoresce at 440 nanometers when excited at 360 nanometers. Coloration in the lenses of diurnally active animals (including man) may be caused by this reaction, and senile cataracts may result. Such changes in many other proteins (as in the skin and retina) could lead to more serious consequences.

Characterization of a cell-lethal product from the photooxidation of tryptophan: hydrogen peroxide.

Near-ultraviolet (300 to 400 nanometers) irradiation of saturated, oxygenated solutions of tryptophan in the absence of added sensitizer gives rise to substances that have various biological effects on isolated cells, including mutagenicity and selective lethality to recombination-deficient bacterial mutants. One of these biologically active products has been identified as H2O2, on the basis of spectrometric, chromatographic, chemical, and biological properties. Now H2O2 has been shown to account for the biological activities mentioned above.

6-Formylindolo[3,2-b]carbazole (FICZ) is a Very Minor Photoproduct of Tryptophan at Biologically Relevant Doses of UVB and Simulated Sunlight.

Exposure to solar UV is at the origin of numerous photodegradation pathways in biomolecules. Tryptophan is readily modified by UVB radiation into ring-opened and oxidized photoproducts. One of them, 6-formylindolo[3,2-b]carbazole (FICZ), has been extensively studied in the recent years because it very efficiently binds to AhR, a major factor in numerous biologic processes, such as metabolism of xenobiotics. Unfortunately, little information is available on the actual yield of FICZ upon exposure to low and biologically relevant doses of UV radiation. In the present work, we used a sensitive and specific HPLC-tandem mass spectrometry assay to quantify a series of photoproducts induced by UVB and simulated sunlight (SSL) in solutions of tryptophan. FICZ represented only a minute amount of the photoproducts (0.02 and 0.03%, respectively). Experiments were repeated in culture medium where the yield of FICZ was also found to be very low, even when Trp was added. Last, no FICZ could be detected in cytosolic fractions of cultured cells exposed to SSL. Altogether, the present results show that FICZ is a very minor photoproduct and that it cannot be considered the only endogenous photoproduct responsible for the induction of AhR-dependent responses in UV-irradiated cells.

Implications of tryptophan photoproduct FICZ in oxidative stress and terminal differentiation of keratinocytes.

Ultraviolet B (UVB) irradiation activates aryl hydrocarbon receptor (AHR), generates reactive oxygen species (ROS) and mediates photocarcinogenesis and photoaging. 6-Formylindolo[3,2-b]carbazole (FICZ) is a tryptophan photoproduct generated by UVB exposure. FICZ exhibits similar biological effects to UVB, including AHR ligation and ROS production. FICZ also acts as a potent photosensitizer for UVA and the production of ROS is synergistically augmented in the simultaneous presence of FICZ and UVA. In contrast, FICZ upregulates the expression of terminal differentiation molecules such as filaggrin and loricrin via AHR. In parallel with this, the administration of FICZ inhibits skin inflammation in a murine psoriasis and dermatitis model. In this article, we summarize the harmful and beneficial aspects of FICZ in skin pathology.

Formation and spectroscopy of a tryptophan radical containing peptide in the gas phase.

A doubly deprotonated tryptophan containing peptide was electrosprayed and isolated in an ion trap. UV excitation on this peptide leads to electron detachment and to the formation of an indolyl radical. The photogenerated radical was fragmented by a second laser. The visible spectrum of the gas-phase neutral tryptophan radical containing peptide has been recorded and constitutes a benchmark for calculations and optical measurements.

Photodynamic efficacy of chlorin p6: a pH dependent study in aqueous and lipid environment.

Photodynamic efficacy of chlorin p6, a potential candidate of photodynamic therapy (PDT), has been studied at pH 5.0, 6.0 and 7.6 in aqueous and lipid environment. Increased chlorin p6 mediated photodynamic bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), a measure of singlet oxygen yield, was obtained at higher pH. Rate of photodynamic bleaching of RNO was also higher at higher pH and the rate decreased with lowering in pH of irradiated solution. Photodynamic oxidation of tryptophan was also found to be higher at higher pH. Diminished oxidation of RNO was obtained with decrease in pH of irradiated solution. Both, RNO bleaching and tryptophan oxidation was significantly reduced by sodium azide, a known quencher of singlet oxygen. At lower pH, chlorin p6 mediated photodynamic malondialdehyde (MDA) and lipid hydroperoxide formation in egg lecithin liposome was higher. At higher pH chlorin p6 was found to be photodynamically more effective in aqueous environment whereas at lower pH chlorin p6 was photodynamically more effective in hydrophobic environment.

Impact of tryptophan on the formation of TCNM in the process of UV/chlorine disinfection.

Low-pressure (LP) UV treatment after chlorine disinfection was associated with enhanced formation of trichloronitromethane (TCNM), a halonitromethane disinfection by-product (DBP), due to the chlorination of tryptophan. Evidence was found that the concentration of TCNM from tryptophan increased quickly to the maximum for the first instance. Moreover, the increase of TCNM under UV exceeded 10 times than under dark. Then, it was found to have an obvious decrease in the formation of TCNM, even finally hardly disappear. In order to elucidate reasons for this phenomenon, the effects of light intensity, initial tryptophan concentration, free chlorine concentration, pH, and tert-butanol (TBA) on the formation of TCNM were investigated under UV/chlorine treatment. Finally, the effects of tryptophan on the formation of TCNM and the direct photodegradation of TCNM under LP UV irradiation were studied for analyzing the possible pathways of TCNM formation from amino acid. Since amino acids are very common in water sources, further research into chemical oxidation of these species by LP UV and chlorine is recommended. It can help us to find the precursors of TCNM formation and reduce the risk of TCNM formation for drinking water and wastewater utilities.

Impact of a single, short morning bright light exposure on tryptophan pathways and visuo- and sensorimotor performance: a crossover study.

BACKGROUND: Bright light (BL) has been shown to be effective in enhancing both cognitive and physical performances. Alterations in nighttime melatonin levels have also been observed. However, evaluations of light-induced changes in the preceding biochemical processes are absent. Therefore, the impact of a single morning BL exposure on sensorimotor and visuomotor performance, as well as tryptophan (trp) and trp metabolites, was evaluated in this study. METHODS: In a crossover design, 33 healthy volunteers were randomly exposed to 30 min of < 150 lx at eye level (office light, OL) and 5000 lx at eye level (bright light, BL) of 6500 K in the morning hours. Trp, sulfatoxymelatonin (aMT6s), and kynurenine (kyn) courses over the morning hours were analyzed, and changes in sensori- and visuomotor measures were examined. RESULTS: Motoric performance increased in both setups, independent of light intensity. aMT6s and kyn decreased equally under both lighting conditions. Trp levels decreased from a mean (95% confidence interval) of 82.0 (77.2-86.9) to 66.5 (62.5-70.1) in the OL setup only. CONCLUSION: These data suggest that BL in the morning hours has a limited effect on visuo- and sensorimotor performance. Nevertheless, trp degradation pathways in the morning show diverse courses after OL and BL exposure. This suggests that trp courses can potentially be altered by BL exposure.

Dissecting Nanosecond Dynamics in Membrane Proteins with Dipolar Relaxation upon Tryptophan Photoexcitation.

The structural dynamics of proteins on the nanosecond time scale can be probed with dipolar relaxation in response to photoexcitation of intrinsic tryptophan (Trp) residues. For membrane proteins, however, the complexity due to overlapping contributions from the protein itself, the membrane mimic, and the aqueous solvent impairs detailed analysis and interpretation. To disentangle these contributions, we measured time-resolved emission spectra of Trp in the protein Mistic in detergent micelles of various polarities. By comparison with Trp analogues in water and micelles, we could dissect the contributions from hydration, micelle, and protein matrix to dipolar relaxation on the nanosecond time scale. Our results demonstrate that ultrafast, subnanosecond relaxation reports on the extent of Trp shielding from water, with micelle and protein moieties making additive contributions. By contrast, relaxation in the low nanosecond regime is due to dipolar rearrangement of micelle and protein moieties upon photoexcitation, thereby probing conformational dynamics around the intrinsic fluorophore.

An endogenous tryptophan photo-product, FICZ, is potentially involved in photo-aging by reducing TGF-ß-regulated collagen homeostasis.

BACKGROUND: Persistent ultraviolet (UV) radiation in the form of sunlight causes photo-aging of the skin by reducing the production of type I collagen, the major constituent of the extracellular matrix of the dermis. Transforming growth factor (TGF)-ß transforms dermal fibroblasts into α2-smooth muscle actin (ACTA2)-expressing myofibroblasts. Myofibroblasts produce a precursor form of type I collagen, type I procollagen (collagen I), consisting of pro-alpha1 (produced by the COL1A1 gene) and pro-alpha2 chains (produced by the COL1A2 gene). Smad2/3 is a key downstream molecule of TGF-ß signaling. The mechanisms through which UV inhibits collagen I synthesis are not fully understood. 6-Formylindolo[3,2-b]carbazole (FICZ) is an endogenous tryptophan photo-metabolite generated by UV irradiation. FICZ is well known as a high-affinity ligand for aryl hydrocarbon receptor (AHR). However, the physiological roles of FICZ in photo-aging have yet to be addressed. OBJECTIVE: To evaluate the effects of FICZ on the TGF-ß-mediated ACTA2 and collagen I expression in normal human dermal fibroblasts (NHDFs). METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine the expression of ACTA2, COL1A1, and COL1A2 in NHDFs with or without FICZ and TGF-ß. The phosphorylated Smad2/3 (pSmad2/3) protein levels in cytoplasmic or nuclear portions were investigated by western blot analysis. Immunofluorescence staining was conducted to evaluate pSmad2/3 localization, and F-actin staining with phalloidin was performed to visualize actin polymerization in myofibroblasts. The actions of FICZ on the TGF-ß-mediated collagen I expression and nuclear translocation of pSmad2/3 were analyzed in the presence of selective AHR antagonists or in AHR-knockdown NHDFs. RESULTS: We found that FICZ significantly inhibited the TGF-ß-induced upregulation of mRNA and protein levels of ACTA2 and collagen I and actin polymerization in myofibroblasts. FICZ did not disturb the phosphorylation of Smad2/3. Notably, FICZ reduced the expression of pSmad2/3 in the nucleus, while it increased that in the cytoplasm, suggesting that it inhibits the nuclear translocation of pSmad2/3 induced by TGF-ß. The inhibitory actions of FICZ on the TGF-ß-mediated collagen I expression and nuclear translocation of pSmad2/3 were independent of AHR signaling. Another endogenous AHR agonist, kynurenine, also inhibited the TGF-ß-mediated ACTA2 and collagen I upregulation in NHDFs in an AHR-independent manner; however, its effects were insignificant in comparison with those of FICZ. CONCLUSIONS: These findings suggest that the endogenous photo-product FICZ may be a key chromophore that involves in photo-aging. Downregulation of FICZ signaling is thus a potential strategy to protect against photo-aging.