Blue light-induced lipid oxidation and the antioxidant property of hypotaurine: evaluation via measuring ultraweak photon emission

The effects of blue light on human body have attracted attention. The human skin in contact with the outside environment is often exposed to blue light, and the effects of this exposure remain to be fully determined. Therefore, in this study, we investigated the effect of blue light, at the intensity typically found in sunlight, on lipids in the skin from an oxidation perspective. Peroxide value (POV) and ultraweak photon emission (UPE) measurements were conducted to evaluate lipid oxidation. Our results confirmed that blue light irradiation induced lipid oxidation, similar to ultraviolet A (UVA) irradiation. Also, the effects of various reagents on the blue light-induced UPE were evaluated; however, the results differed from those of the DPPH radical-scavenging ability. We speculated that this is due to the difference in the evaluation principle; nevertheless, among reagents, hypotaurine not only showed a high antioxidant effect but was also more effective against blue light-induced oxidation than UVA. Based on the difference in the antioxidant effect of the lipid sample in this study, the oxidation reaction induced by blue light may be different from the UVA-induced reaction. Our study provides new insights into the effects of blue light on lipids in the human skin, thereby promoting research regarding photooxidation.


Introduction
Light affects cells and biomolecules in living organisms, potentially influencing their physiological functions [1]. Light irradiation exerts both positive and negative effects on animals and plants [2,3]. Light of different wavelengths is used in medical treatment [4] as well as to regulate plant growth [3]. Several studies on blue light with relatively strong intensity in the visible light spectrum have been reported. Blue light affects the circadian rhythm and inhibits melatonin production [5]. Blue light interacts with opsin-3, thereby regulating melanogenesis in melanocytes [6]. Blue light also induces oxidative stress [7], an underlying cause of cell dysfunction and diseases [8]. The human 1 3 skin is constantly exposed to various sources of blue light, including sunlight, devices, and interior illumination; hence it is important to understand the effect of chronic exposure to blue light. The blue light in sunlight is particularly intense [9]. Therefore, in this study, we explored the effect of blue light irradiation at the intensity typically found in sunlight to investigate the effect of blue light exposure on lipids in the skin.
Numerous components can be oxidized in the body; for instance, lipids can be converted to lipid peroxides, which participate in various pathological conditions [10]. As lipids are present in cell membranes and between cells, considerably influencing biological functions [11], it is important to clarify the factors that trigger lipid oxidation. Ultraviolet (UV) light induces oxidative stress in the skin [12], and we previously proposed that some biomolecules in the skin undergo oxidation under ultraviolet A (UVA) irradiation [13]. Similarly, blue light causes oxidation. However, only a few studies have focused on blue light exposure at the intensity to which humans are typically exposed in daily life and investigated the physiological effects. Of note, the intensities of blue light emitted from the sun and that emitted from devices are reportedly different [9]. Therefore, in this study, we investigated the possibility of blue light-induced lipid oxidation caused by daily exposure to sunlight by measuring the intensity of blue light in the sunlight. In addition, we assessed the antioxidants that might be effective against blue light-induced oxidation. Peroxide value (POV) [14] and ultraweak photon emission (UPE) [15] measurements were performed to evaluate lipid oxidation. In particular, we evaluated lipid oxidation and antioxidant effects via UPE measurement.
UPE measurement has been applied in various research fields, including research on plants [16], insects [17], animals [18], and humans [19]; furthermore, numerous studies have demonstrated the usefulness of UPE measurement. These studies have mostly reported the relationship between UPE intensity and reactive oxygen species (ROS). In addition, as the approach is noninvasive, UPE measurement is expected to be a diagnostic method for diseases related to oxidative stress. Generally, UPE is observed in the oxidation and peroxidation processes of biological substances by reactive oxygen species (ROS) and free radicals [20]. In lipid peroxidation, ROS act as initiators, and excited carbonyl molecules and excited singlet oxygen are generated. It is presumed that these excited molecular species and biological substances excited by energy transfer become luminescent species. Although UPE is an extremely weak spontaneous luminescence (approximately 10 -16 W/cm 2 [10 3 photon/s/ cm 2 ] or less), it can be detected using a photomultiplier tube (PMT) or a high-sensitivity charge-coupled device (CCD) camera without labelling. Based on these mechanisms, UPE have been applied to evaluate oxidative stress [21,22]. In this study, we applied UPE measurement to evaluate the oxidation of lipid samples and that in the skin tissue.

Spectral analysis of sunlight and light from the screens of electronic devices
The UV-visible wavelength spectrum was obtained using a spectrometer QEPro (Built-in grating HC-1) with an optical fiber QP450-1-XSR (Ocean Insight, Orlando, FL, USA). Visible-IR wavelength spectra were obtained using a spectrometer NIRQuest512 with an optical fiber QP1000-2-VIS/ NIR (Ocean Insight). The sunlight spectrum was obtained in August (Yokohama, Japan). For the device screen, the spectrometer sensor was placed 0 or 10 cm from the screen. A white image was projected onto the device screen, and the screen spectrum was obtained by maximizing illumination without attachment for the screen. Blue light intensities (400-500 and 400-450 nm) were calculated from the spectra of the light emitted from sunlight and several devices.

Lipid samples and skin tissue used to evaluate oxidation
Linoleic acid (1st Grade, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), squalene (Special Grade, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and oleic acid (O0180, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were selected as lipid samples. Human skin tissue derived from the abdomen of a 42-year-old Caucasian female was purchased from Biopredic International (Rennes, France) via KAC (Kyoto, Japan).

Blue light and UVA irradiation
A 430 nm LED (CL-H1-430-9-1-B, peak at 430 nm with full width at half maximum of 17 nm, Asahi Spectra Co., Ltd., Tokyo, Japan) was selected as the blue light source. The intensity of blue light was measured using a Hioki 3664 optical power meter with a 9742 optical sensor (Hioki E.E. Corporation, Nagano, Japan). Torex FL20SBL/DMR (Toshiba Medical Supply, Tokyo, Japan) was used as the UVA source. UVA intensity was measured using a Dermaray UV-meter DMR-UV-M-2 with a Dermaray UV Detector DMR-UV-ABBNB-2 (Gigahertz-Optik GmbH, Germany).

Measurement of POV
Thirteen milliliters of lipid reagents (linoleic acid and squalene) was placed in a 50 mL glass beaker and irradiated with UVA or blue light under air exposure. Thereafter, samples were subjected to POV measurement. The procedure for measuring the POV was as follows: 30 mL of a mixed solution of chloroform and acetic acid (chloroform:acetic acid = 2:3) was added to 5 g of the test sample and dissolved. Nitrogen substitution was performed, a saturated potassium iodide solution (0.5 mL) was added, and the mixture was allowed to react in the dark for 5 min. After the reaction, water (30 mL) was added and mixed thoroughly. After that, titration (indicator: 1% starch solution) was performed using 0.01 mol/L sodium thiosulfate standard solution, and the POV (meg/kg) was determined using the following formula: where V is the volume of titration (mL), F is the factor of sodium thiosulfate solution (0.01 mol/L), and W is the sample weight (g).

UPE photon-counting system
UPE intensity was measured using a photon-counting system [22][23][24] as a special-order device manufactured by Tohoku Electronic Industrial Co., Ltd. (Sendai, Japan), which was used for the evaluation of oxidative stress. The photoncounting system was equipped with a photomultiplier tube (PMT; R2257P; Hamamatsu Photonics K.K., Japan; spectral response 300-900 nm with a peak at 600 nm), a cooling system, and a dark chamber. The PMT was cooled to approximately − 35 °C to improve the signal-to-noise (S/N) ratio and reduce the dark current. The samples were transferred to a quartz cell for measurement, set in a dark chamber of a photon-counting system, and UPE was detected using a PMT placed under the quartz cell. The samples were transferred to the quartz cell after light irradiation to avoid light exposure to the quartz cell. The measurement was started 60 s after the light exposure. The UPE photon count was recorded every second, and the integrated value of the measured values for 300 s was calculated to evaluate oxidative stress.

UPE imaging system
UPE images for the evaluation of oxidative stress were obtained using a UPE imaging system designed by referring to previous research [17,25]; the imaging system is a special-order device manufactured by Tohoku Electronic Industrial Co., Ltd. (Sendai, Japan). Samples were placed on a stage in the dark chamber of the imaging system. UPE images were continuously captured with 10-min exposures using a cooled CCD camera (SI 850 s; Spectral Instruments Inc., USA, equipped with e2v CCD42-40) with a Nikkor lens (50 mm f/1.2) and PK-13 auto extension ring (Nikon Corporation, Tokyo, Japan). The CCD operating temperature was − 90 °C. Imaging was performed on a surface area of 43 mm × 43 mm. In this experiment, the CCD was operated in the 16 × 16 binning modes, and the actual pixel number was 128 × 128. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for UPE image analysis.

Measurement of blue light-or UVA-induced UPE in lipid samples
After irradiating 3 mL of linoleic acid with blue light (1-10 mW/cm 2 , 10 min) or UVA (0.36-3.9 mW/cm 2 , 10 min) under air exposure, the linoleic acid was transferred to a quartz cell to avoid luminescence from other substances, and UPE measurements were started immediately. The interval from the end of irradiation to the start of UPE measurement was set to 60 s. After irradiation, the UPE intensity for 300 s from the start of measurement was integrated. The integrated intensity of the non-irradiated control was subtracted to obtain the actual UPE intensity induced by blue light or UVA.

Evaluation of antioxidant effect using UPE
The antioxidant effects against the oxidation of linoleic and oleic acids were evaluated using the UPE photon-counting system. The following reagents were selected to evaluate their antioxidant effects on blue light-and UV-induced oxidation: l(+)-ascorbic acid (Guaranteed Reagent, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), thiotaurine (2-aminoethanethiosulfonic acid, Sogo Pharmaceutical Co., Ltd., Tokyo, Japan), hypotaurine (2-aminoethanesulfinic acid, Sogo Pharmaceutical Co., Ltd., Tokyo, Japan), calcium pantetheine sulfonate (calcium d-pantetheine-S-sulfonate 70 [PSS-70], 70% aq., Sogo Pharmaceutical Co., Ltd., Tokyo, Japan), taurine (Qianjiang Yongan Pharmaceutical Co., Ltd., Hubei, China), ascorbyl glucoside (AA2G, Hayashibara Co., Ltd., Okayama, Japan), and l-cysteine (Special Grade, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Test samples consisted of 1 mL of linoleic acid or oleic acid, 0.95 mL of 1,3-butanediol (Special Grade, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and 0.05 mL of an aqueous solution of selected reagents (5%[w/w] aq.) for the evaluation of antioxidant effects. Milli-Q water was used as the control instead of an aqueous solution of the selected reagents. After irradiating the test samples with blue light (6 mW/cm 2 , 10 min) or UVA (3.5 mW/cm 2 , 10 min) under air exposure, the test samples were transferred to quartz cells to avoid luminescence from substances other than the test sample, and UPE measurements were started immediately. The interval from the end of irradiation to the start of the UPE measurement was set to 60 s. After irradiation, the UPE intensity for 300 s from the start of measurement was integrated. The integrated intensity of the non-irradiated control was subtracted to obtain the actual UPE intensity induced by blue light or UVA. The UPE suppression rate of each reagent was calculated as follows: For the evaluation using UPE imaging system, test samples consisted of 250 μL of linoleic acid, 237.5 μL of 1,3-butanediol, and 12.5 μL of an aqueous solution of the selected reagents (5% [w/w] aq.). Milli-Q water was used as the control instead of an aqueous solution of the selected reagents. After irradiation with blue light (5.4 mW/cm 2 , 10 min), the test samples (300 μL) were transferred to each well of a black flat-bottom 96-well microplate to avoid luminescence from substances other than the test sample, and UPE images were captured immediately (exposure time was 10 min).
scavenging rate (%) was calculated following the kit manufacturer's instructions.

Statistical analysis
Tukey-Kramer test was used for POV measurement. Student's t test and paired t test were used for UPE measurement. The change/difference was considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001. Statistical analysis was performed using Statcel-the Useful Addin Forms on Excel-4th ed. Data are presented as mean ± standard deviation (SD).

Blue light of sunlight and the device screens
The spectra of sunlight (August) and the light of each device screen were obtained using a spectrometer, and blue light intensities in the wavelength ranges of 400-500 nm and 400-450 nm were calculated. The blue light in the sunlight was a few orders of magnitude stronger than that of the devices ( Table 1). The light spectra of sunlight and several device screens were obtained using a spectrometer. Blue light intensities (400-500 nm, 400-450 nm) were calculated from the light spectra. The sensor of the spectrometer was placed at 0 or 10 cm from the device screen.

Peroxidation induced by blue light or UVA
Linoleic acid and squalene used as lipid samples were irradiated with UVA or blue light, and the changes in POV were  investigated. UVA induced an increase in the POV, and we confirmed that UVA oxidized linoleic acid and squalene (Fig. 1a). In addition, blue light increased the POV, similar to UVA, and intensity-dependent changes were confirmed. Although the irradiation intensity was different, it was confirmed that blue light also induced the peroxidation of linoleic acid and squalene. After irradiation with the same blue light or UVA intensity, UVA showed a higher ability to oxidize linoleic acid and squalene than blue light (Fig. 1b).

Change in UPE intensity induced by blue light or UVA
The UPE of linoleic acid after blue light or UVA irradiation was measured using the UPE photon-counting system. Photon counts were measured every 1 s; Fig. 2a shows an example of the measured photon counts. The photon count was increased by blue light irradiation, and the count declined with time. UVA-induced UPE photon counts showed similar attenuation. The UPE of squalene was not detected using the UPE photon-counting system. The integrated value of UPE for 300 s was calculated, and the UPE before irradiation was subtracted from the UPE after light irradiation to obtain the light-induced UPE. The results of blue light or UVA-induced UPE (300 s) are shown in Fig. 2b, c. Blue light induced an increase in UPE of linoleic acid in a dose-dependent manner, and the tendency was the same as that of UVA irradiation. However, the ability of UVA to increase UPE was higher than that of blue light.

Effect of selected reagents against blue light or UVA-induced UPE
Test samples containing linoleic acid, 1,3-butanediol, and an aqueous solution of the reagents were irradiated with blue light, and the UPE photon count was measured. The intensity of UPE for 300 s induced by blue light irradiation and the suppression rate were calculated. Figure 3a shows that the blue light-induced UPE was suppressed by l-ascorbic acid, thiotaurine, hypotaurine, and PSS-70; in particular, thiotaurine and hypotaurine were confirmed to have high inhibitory effects against UPE. Conversely, ascorbyl glucoside, taurine, and l-cysteine showed almost no inhibitory effects. Reagents with a suppression effect against the blue light-induced UPE were also effective against UVA-induced UPE (Fig. 3b). In addition, thiotaurine and hypotaurine showed characteristic effects, indicating that these reagents were more effective against blue light-induced UPE than UVA-induced UPE (Fig. 3c). Additionally, the effects of reagents against blue light-induced UPE were visualized using the UPE imaging system (Fig. 4). Hypotaurine suppressed blue light-induced UPE and it was the same as the result using the UPE photon-counting method. These evaluations confirmed that hypotaurine had the highest effect against blue light-and UVA-induced UPE. Although the test samples contained 1,3-butanediol, no blue light-induced UPE and UVA-induced UPE of 1,3-butanediol was observed. The effect of hypotaurine on oleic acid was investigated to evaluate the antioxidant effect of hypotaurine. Similar to linoleic acid, oleic acid showed blue light-induced UPE and UVA-induced UPE (Fig. 5a). Hypotaurine was also effective against blue light-and UVA-induced UPE. As on UPE of linoleic acid, it showed a significantly higher suppression effect on blue light-induced UPE than UVA-induced UPE (Fig. 5b).

DPPH radical scavenging by reagents
The DPPH radical scavenging activity of the reagents used in the UPE evaluation was evaluated. This test confirmed the high DPPH radical scavenging ability of l-ascorbic acid, ascorbyl glucoside, hypotaurine, and l-cysteine (Fig. 6). Thiotaurine was effective only at high concentrations. However, no noticeable DPPH radical scavenging effect was observed for taurine and PSS-70. l-Ascorbic acid and hypotaurine, which affected blue light-and UVA-induced UPE, showed a high DPPH radical scavenging effect. Still, thiotaurine and PSS-70 did not show the DPPH radical scavenging effect, as expected from the UPE results. In contrast, although ascorbyl glucoside and l-cysteine were ineffective against blue light-or UVA-induced UPE, they showed DPPH radical scavenging effects. Taurine showed no effect in either evaluation.

Discussion
Humans are routinely exposed to the light of several wavelengths in daily life, among which UV is known to affect living beings. UV exposure can lead to oxidative stress in Fig. 4 Imaging of blue lightinduced UPE suppressed by reagents. Test samples containing linoleic acid, 1,3-butanediol and aqueous solution of the reagents were irradiated with blue light (5.4 mW/cm 2 , 10 min). The photograph on the left was taken under weak illumination and corresponds to UPE image on the right. UPE image on the right was captured using a CCD camera with a 10 min exposure. The color scale indicates the signal intensity from 0 (black) to 27 (white) humans, which is associated with various pathophysiological conditions, including skin problems [27]. Additionally, blue light in visible light is associated with ROS generation [7], but the effects of blue light on human skin must be carefully investigated. Blue light in sunlight is a few orders of magnitude stronger than that emitted by several electronic devices, as shown in our results and previous research [9]. In this study, we focused on the impact of blue light at the intensity typically found in sunlight on lipids and determined whether blue light oxidizes lipids. We found that blue light caused lipid oxidation, although the degree of oxidation was weaker than that induced by UVA, according to POV and UPE measurements. Furthermore, our results suggest that blue light in sunlight can exert oxidative stress on living beings. For instance, as evidenced by imaging, UPE from the human skin tissue increased upon blue light exposure. It was observed that blue light induces oxidative stress in the human skin (Fig. 7). Lipid peroxide adversely affects cell function and the skin [28]. Lipid peroxides are mediators of diseases [11], and the antioxidant system can be affected by protein adducts with lipid peroxidation products [29]. The addition of linoleic acid hydroperoxide to human skin fibroblasts affects the production of matrix metalloproteinases involved in collagen metabolism [30]. Peroxides of squalene and oleic acid enhance comedo formation in rabbit skin [31]. Thus, lipid peroxidation may affect the living body following oxidation; hence, it is important to elucidate the cause of oxidation and develop an antioxidant method.
The POV and UPE measurements were used as oxidation indicators in this study, and the antioxidant effects were assessed by UPE measurement, which is a more sensitive method. Among the selected reagents, thiotaurine and hypotaurine, which are sulfur-containing amino acid derivatives [32], strongly suppressed blue light-induced UPE. These results are consistent with those of previous studies showing that thiotaurine and hypotaurine exert antioxidative effects [33][34][35]. In addition, the inhibitory effects of PSS-70, a sulfur-containing reagent, against UPE were also confirmed; however, these effects were relatively weak. Conversely, taurine, a sulfur-containing amino acid derivative, and l-cysteine, a sulfur-containing amino acid, did not exert an inhibitory effect even though they are known as antioxidants [36,37]. l-Ascorbic acid and its derivative ascorbyl glucoside showed different results: only l-ascorbic acid inhibited the increase in UPE. Furthermore, the DPPH radical scavenging rate is not consistent with the results of the UPE suppression effect. These differences suggest that UPE is derived from non-radical and other radical species.
Unexpectedly, thiotaurine and hypotaurine showed stronger suppression effects on blue light-induced UPE than on UVA-induced UPE, suggesting different oxidation mechanisms between UVA and blue light. As hypotaurine showed the same result as oleic acid, other unsaturated fatty acids may show the same tendency. As we confirmed that hypotaurine itself does not absorb the UPE generated from lipid samples (data not shown), it can be considered that hypotaurine suppresses the UPE generation induced by irradiation. Additionally, only hypotaurine had a large DPPH radical scavenging ability. This result suggests that hypotaurine has antioxidant effects on both non-radical and radical species.
The oxidation of lipids in this study was considered autoxidation (radical chain reaction) [38]. Irradiation with UVA or blue light in the presence of oxygen abstracts hydrogen from lipid molecules and produces hydroperoxides via peroxy radicals. POV is an indicator of hydroperoxide, and peroxy radicals produce singlet oxygen and triplet excited carbonyls responsible for UPE [20,39]. UVA or blue light irradiation increased the POV of lipid samples as well as UPE. We presumed that this results from hydroperoxide formation by irradiation with either UVA or blue light. Considering the difference in reactivity between blue light-and UVA-induced UPE confirmed in this study, we suggest that thiotaurine and hypotaurine are more effective against initial oxidation.
We propose that blue light affects the oxidation phenomenon in our daily lives and further suggest that the underlying mechanism differs from that of UVA-related oxidation because of the effectiveness of antioxidant effects. It has been reported that there is a difference in mechanism between photo-and thermal-oxidation of polyethylene, even in the same oxidation phenomenon [40]. Therefore, the difference between UV-and blue lightinduced oxidation must be investigated in more detail in the future. A study has suggested differences in the reactivity of blue light and UVA in the skin [7].
UV-induced oxidative stress is associated with eye health [41]. As the relationship between cataracts in humans and lipid peroxides has been characterized in previous studies [42,43], data regarding light oxidation can contribute to the field of ophthalmology. In addition, hypotaurine may be applied as an antioxidant and added to foods that are exposed to light and undergo oxidative deterioration [44]. As hypotaurine is a metabolite of sulfur-containing amino acids in mammals [45,46], its effects warrant further investigations.
In conclusion, our study showed that blue light of the same intensity as in sunlight oxidizes lipids. In addition, there is the possibility that the photooxidation mechanisms Fig. 7 Imaging of blue light-induced UPE in the human skin tissue. Human skin tissue (Caucasian) was irradiated with blue light (6 mW/cm 2 , 10 min) from the stratum corneum side. Subcutaneous tissue was removed prior to the experiment. a The photograph on the left was taken under weak illumination and corresponds to the UPE image on the right. The UPE image on the right was captured using a cooled CCD camera with a 10 min exposure. The color scale indicates the signal intensity from 0 (black) to 75 (white). b UPE intensity of the skin tissue determined from UPE imaging data. Data are presented as mean ± SD, N = 4; Paired t test, ***p < 0.001 1 3 caused by blue light and UVA may differ, warranting further investigation to clarify this difference. Additionally, we propose that hypotaurine is more effective against blue light-induced lipid oxidation. Our study provides new insights into the effects of blue light, thereby advancing photochemistry and photobiology research.