Hung-Chi Tua, Meng-Yun Linb, Chia-Yang Linb, Tsun-Hsien Hsiaoa, Zhi-Hong Wenc, Bing-Hung Chend,e,f,g,∗∗, Tzu-Fun Fua,b,∗
aThe Institute of Basic Medical Sciences, National Cheng Kung University, College of Medicine, Tainan, Taiwan
bDepartment of Medical Laboratory Science and Biotechnology, National Cheng Kung University, College of Medicine, Tainan, Taiwan
cDepartment of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan
dDepartment of Biotechnology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
eDepartment of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
fCenters for Biomarkers and Biotech Drugs, Kaohsiung Medical University, Kaohsiung, Taiwan
gDepartment of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan
A R T I C L E I N F O
Keywords:
UV-inflicted damage UV protection Folate
5-Formyltetrahydrofolate Zebrafish
A B S T R A C T
Ultraviolet (UV) is an omnipresent environmental carcinogen transmitted by sunlight. Excessive UV irradiation has been correlated to an increased risk of skin cancers. UVB, the most mutagenic component among the three UV constituents, causes damage mainly through inducing DNA damage and oxidative stress. Therefore, strate-gies or nutrients that strengthen an individual's resistance to UV-inflicted harmful effects shall be beneficial.
Folate is a water-soluble B vitamin essential for nucleotides biosynthesis, and also a strong biological anti-oxidant, hence a micronutrient with potential of modulating individual's vulnerability to UV exposure. In this study, we investigated the impact of folate status on UV sensitivity and the protective activity of folate sup-plementation using a zebrafish model. Elevated reactive oxygen species (ROS) level and morphological injury were observed in the larvae exposed to UVB, which were readily rescued by supplementing with folic acid, 5-formyltetrahydrofolate (5-CHO-THF) and N-acetyl-L-cysteine (NAC). The UVB-inflicted abnormalities and mortality were worsened in Tg(hsp:EGFP-γGH) larvae displaying folate deficiency. Intriguingly, only supple-mentation with 5-CHO-THF, as opposed to folic acid, offered significant and consistent protection against UVB-inflicted oxidative damage in the folate-deficient larvae. We concluded that the intrinsic folate status correlates with the vulnerability to UVB-induced damage in zebrafish larvae. In addition, 5-CHO-THF surpassed both folic acid and NAC in preventing UVB-inflicted oxidative stress and injury in our current experimental zebrafish model.
1. Introduction
UV is an environmental carcinogen that people are exposed to on a daily basis. Excessive UV exposure has been shown to positively cor-relate with the occurrence of a wide spectrum of skin pathologies, in-cluding inflammation, pigmentation, wrinkling and skin cancer, the most common malignancy in Western society (Apalla et al., 2017;
D'Orazio et al., 2013). Global Cancer Observatory (GCO) reported that there are estimated 1.3 million skin cancer cases occurring and causing 125 thousand deaths in 2018 (Bray et al., 2018). Photoprotective
measures, including limited sun exposure during peak UV radiation hours, wearing protective clothing and applying sunscreens, are re-commended to avoid UV damage. However, completely blocking the exposure to solar UV is impractical and unsuitable since proper sunlight exposure is essential for maintaining health, especially through vitamin D synthesis (Lee et al., 2013;Wright and Weller, 2015). The reported allergic responses and phototoxicity caused by the sunscreen in-gredients also raise concerns regarding the safety and effectiveness of sunscreen applications (Foley et al., 1993;Gonzalez et al., 2008;Nash, 2006). Therefore, the exploration of novel strategies and nutrients
https://doi.org/10.1016/j.ecoenv.2019.109380
Received 18 May 2019; Received in revised form 19 June 2019; Accepted 21 June 2019
∗Corresponding author. Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, College of Medicine, No.1, University Road, Tainan, 701, Taiwan.
∗∗Corresponding author. Department of Biotechnology, Kaohsiung Medical University, College of Life Science, No. 100, Shih-Chuan First Road, Kaohsiung, 807, Taiwan.
E-mail addresses:[email protected](B.-H. Chen),tff[email protected](T.-F. Fu).
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capable of intrinsically strengthening an individual's physical capability to resist or accommodate UV-inflicted harmful effects are warranted.
Studies have shown that the UV-inflicted damage are majorly re-lated to impeded genomic integrity and increased oxidative stress (Lee et al., 2013). Sunlight encompasses three different types of UV radia-tion: UVA (315–400 nm), UVB (280–315 nm) and UVC (100–280 nm).
UVC, even though with the highest radiation energy, is absorbed by the ozone in the atmosphere therefore hardly reaches the surface of earth.
Therefore, UVB has been considered as the most mutagenic UV com-ponent, which induces DNA damage by promoting the formation of pyrimidine dimers, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) (Bowden, 2004;Budden and Bowden, 2013;
Schuch et al., 2017). UVB radiation also generates reactive oxygen species (ROS), leading to epidermal damage (Schuch et al., 2017).
Therefore, compounds/nutrients supporting DNA repair and with anti-oxidative activity are of benefit to photoprotection against UV
exposure.
Folate, also known as vitamin B9, is enriched in leafy green vege-tables and holds the potential of modulating individual's susceptibility to UV exposure (Williams et al., 2012;Williams and Jacobson, 2010).
Folate is also included in most multivitamin and nutrient supplements available in the food/drug stores around the corner. Acting as an one-carbon carrier in one-one-carbon metabolism (OCM), folate participates in the biosynthesis of purines and thymidylate, and is crucial for main-taining genome stability (Fox and Stover, 2008). Folate provides its one-carbon unit for S-adenosylmethionine (SAM) formation. SAM is the methyl donor for most intracellular methylation reactions including DNA and RNA methylation, hence an important factor of epigenetic regulation. Folate itself is also a strong antioxidant (Stanger and Wonisch, 2012). In addition, folate-mediated OCM generates approxi-mately half of the intracellular NADPH, contributing significantly to cellular redox balance and reductive biosynthesis (Fan et al., 2014). The Abbreviations
UV Ultraviolet
ROS Reactive oxygen species OCM One-carbon metabolism SAM S-adenosylmethionine NAC N-acetyl-L-cysteine THF Tetrahydrofolate
5-CHO-THF 5-Formyltetrahydrofolate FA Folic acid
H2DCFDA 2′,7′-Dichlorofluorescin diacetate
TAC Total antioxidant capacity
WT Wild-type
CTL Control
MFD Mild folate deficiency SFD Severe folate deficiency FD Folate deficiency Hpf Hour-post-fertilization Dpf Day-post-fertilization Hpuv Hour-post-UVB exposure Dpuv Day-post-UVB exposure
Fig. 1. Structures of folate adducts. (A) Tetrahydrofolate, a natural form of folate composed of a fully reduced pterin ring, a para-aminobenzoyl group (PABA) and a γ-linked polyglutamate tail with up to nine glutamate (Glu) residues. (B) Reduced folate adducts are named based on the bound one-carbon moieties at N5 or/and N10 position. (C) Folic acid, the synthetic form of folate with an oxidized pterin ring and a single glutamate residue (Glu). (D) 5-formyltetrahydrofolate (5-CHO-THF), a relatively stable form of reduced folate used in the current study, carries a 5-formyl group at the N5 position of pterin ring. Arrows in (A) and (D) indicate the cleavage site ofγ-glutamylhydrolase (γGH).
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multifaceted activities of folate empower this water-soluble vitamin a crucial role in defending against UV-inflicted damage. Nowadays, folate deficiency (FD) is still common even in well-nourished populations (Bailey et al., 2015). The persistence of FD often leads to ultimate ill-ness, especially in people with mild deficiency while concomitantly lacking self-awareness on their own folate status. Considering the photoprotective potential of folate and easy access to folate supplement for general public, this vitamin might serve as a convenient, efficient and economical alternative for preventing UV-related illness.
In general, folate refers to a mixture of pterins with distinctive structures. Folate is composed of a pteridine ring, a para-aminobenzoyl group (PABA) and aγ-linked polyglutamate tail of five to nine gluta-mate (Glu) residues (Fig. 1A). All naturally occurring folate is tetra-hydrofolate (THF), which possesses a fully reduced pterin ring found to be the antioxidant phamacophore of folate and carries one-carbon units at the oxidation levels of formate, formaldehyde and methanol bound to N-5 and/or N-10 positions, forming a large group of one-carbon adducts supporting the biosynthesis of purines, thymidylate, and SAM (Fig. 1B).
Reduced folate is also a radical scavenger and possesses antioxidant activity comparable to vitamin C (Rezk et al., 2003; Stanger and Wonisch, 2012). Contrarily, folic acid, the synthetic folate adduct, is the oxidized form of folate included in fortified food, most over-the-counter vitamin supplements, and cosmetic products (Fig. 1C) (Debowska et al., 2005). Folic acid is biologically inactive. Upon transported into cytosol, folic acid needs to be fully reduced and polyglutamylated to become active. The growing awareness of the connection between many dis-eases and FD has raised the public demand for folate supplementation although the efficacy and associated fundamental mechanism remain elusive. Ample amounts of folic acid are consumed, especially by pregnant women, as a daily nutritional supplement. The benefits of folic acid supplementation and fortification have been well documented, especially for the prevention of fetal neural tube defects (Viswanathan et al., 2017). No immediate cytotoxicity has been reported regarding the excessive supplementation of the most commonly used reduced folate, 5-formyltetrahydrofolate (5-CHO-THF) (Strickland et al., 2013).
Nonetheless, increasing numbers of studies also reported the detri-mental impacts associated with excessive supplementation of the syn-thetic folate (i.e. folic acid), including interfered immunity and in-creased risk for tumorigenesis, raising public concerns on the safety of folic acid supplementation among researchers (Kim, 2006;Sawaengsri et al., 2016; Selhub and Rosenberg, 2016; Troen et al., 2006; Wien et al., 2012). In addition, retrospective cohort studies revealed a pro-spective association between higher folate intake and increased in-cidence of skin cancers (Donnenfeld et al., 2015;Fung et al., 2002;van Dam et al., 2000), making understanding the impact of folate on health, specifically on individual's vulnerability to UV exposure, an indis-pensable prerequisite for the safe and effective use of folate supple-mentation.
In this study, we aim to investigate the influence of folate status and folate supplementation on individual's susceptibility to UV-inflicted damage using zebrafish, a powerful in vivo model emerging during the past few decades for biomedical research, as our experimental ap-proach. With the advantages of high fecundity, quick development, easy observation, easy and low cost maintenance, high genetic simi-larity to human and permeability to small molecules, zebrafish sur-passes most other animal models as an in vivo platform for high-throughput screening (Bambino and Chu, 2017; Chakraborty et al., 2009;Howe et al., 2013;Komoike and Matsuoka, 2016). Zebrafish has also been used in research related to UV-toxicology, with which evi-dence greatly advancing our knowledge in thisfield has been generated (Aksakal and Ciltas, 2018; Almeida et al., 2015; Dong et al., 2007;
Hurem et al., 2018; Torres Nuñez et al., 2012; Yang et al., 2012).
However, studies with zebrafish on the effects of folate, especially FD, for their sensitivity to UV radiation has not been reported. This is likely due to the limitation of proper protocol to induce FD in zebrafish, owing to their characteristics in feeding habits and growing
environment. Previously, we had developed afluorescent transgenic line, Tg(hsp:EGFP-γGH), which displays FD in a stage-, intensity-, and duration-controllable manner upon induction (Kao et al., 2014). The over-expressed recombinantγ-glutamylhydrolase (γGH) converts folate polyglutamates to monoglutamate and facilitates folate exportation out of the cells, resulting in intracellular FD. With this model, the responses of larvae with/without FD and folate supplementation to UVB exposure were examined and discussed.
2. Materials and methods 2.1. Fish line and maintenance
The AB strain zebrafish was purchased from Taiwan Zebrafish Core Facility. The zebrafish transgenic line, Tg(hsp:EGFP-γGH), displaying heat-shock-induced folate deficiency, was developed in our lab as previously described (Kao et al., 2014). The maintenance of zebrafish followed the standard husbandry guide (Westerfield, 2007). Both the uses and experiments performed with the zebrafish animal model were approved by the Affidavit of Approval of Animal Use Protocol of Na-tional Cheng-Kung University (IACUC Approval NO. 106086).
2.2. Materials
5-formyltetrahydrofolate (5-CHO-THF) was purchased from Schircks Laboratories. Folic acid (FA), N-acetyl-L-cysteine (NAC) and 2′,7′-Dichlorofluorescin diacetate (H2DCFDA) were purchased from Sigma-Aldrich (MO, USA). The Total Antioxidant Capacity Assay Kit was purchased from Abcam (Cambridge, UK).
2.3. Induction of folate deficiency
Embryos displaying induced FD were prepared as previously de-scribed (Kao et al., 2014). In brief, embryos generated from hetero-zygous Tg(hsp:EGFP-γGH) transgenic line were heat-shocked at 38–39 °C for 1 h when reaching 9 and 24 h-post-fertilization (hpf).
Embryos were categorized based on theirfluorescence intensity which has been shown to positively correlate with the extent of FD. The in-tensity offluorescence was classified through direct observation under a fluorescence dissecting microscope. Those embryos exhibited sig-nificant green fluorescence under low magnification (16x) were clas-sified into severe folate deficiency group, other embryos showed ob-vious greenfluorescence only under higher magnification (> 32x) were classified into mild folate deficiency group.
2.4. Compound treatment
Compounds, including 5-CHO-THF, FA and NAC, were freshly pre-pared in E3 embryo water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4), added to the wells containing embryos of 10.5 hpf to reach the indicated concentrations. The embryo water with compounds were refreshed every day until the end of observation. Unless otherwise specified, the concentrations used were 500 μM for 5-CHO-THF/FA and 20μM for NAC.
2.5. UV exposure
Larvae at 6 day-post-fertilization (dpf), where functional melano-cytes have developed, were removed from the compound solutions and to a 6-well culture dish containing 3 mL of E3 embryo water per well.
The culture dish was maintained at room temperature while exposed to of UVB radiation (302 nm, 1 J/cm2) in a UVP crosslinker (CL-1000M, Analytik Jena). Larvae were transferred to freshly prepared embryo water with compounds after exposure and assessed visually under a dissecting microscope at 1-day-post-UVB exposure (dpuv) for morpho-logical anomalies. A customized scoring system was employed to
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quantify the UVB-elicited abnormalities in larval tailfin, a region which has been used to evaluate the UV-induced damage in zebrafish pre-viously (Cheng et al., 2014). Score zero represents intact tailfin, one and two represent mild and severe tailfin shrinkage respectively, and three represents disrupted tailfin.
2.6. Reactive oxygen species assay
The reactive oxygen species (ROS) in embryos were detected using 2′,7′-Dihydrodichlorofluorescein diacetate (H2DCFDA) staining. In brief, larvae of 6-dpf were immersed in 100μΜ of H2DCFDA solution for 30 min. After rinsed with embryo water, the greenfluorescent ROS signal was examined under afluorescence dissecting microscope (Ex485 nm/Em560 nm).
2.7. Total antioxidant capacity assay
Larval antioxidant capacity was evaluated with a colorimetric method using the Total Antioxidant Capacity Assay (TAC) Kit following the manufacture's instruction (Abcam. Cambridge, UK). In brief, larvae were homogenized in phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), incubated on ice for 10 min and centrifuged at 4 °C to remove particulates. The supernatant was
collected and incubated with Cu2+working solution for 90 min. For small molecule TAC, the supernatant was diluted 1:1 with protein mask solution to exclude the antioxidant capacity contributed by proteins before incubating with Cu2+ working solution. The reduced copper Cu+generated in the solution, representing larval antioxidant capacity, was quantified with OD570 nmand presented as the equivalent capacity of trolox, a standardized antioxidant.
2.8. Folate measurement
The intracellular folate content and composition of larvae were measured as previously described (Kao et al., 2013). For total folate content, Lactobacillus casei microbiological assay was used with mod-ification (Horne and Patterson, 1988). In brief, Lactobacillus casei grown in Difco™ Folic acid Casei Medium (Becton, Dickinson and company;
MD, USA) at 37 °C in the presence of tested samples was quantified with OD600nm. The content of larval total folate was calculated by inter-polation on the standard growth curve constructed with folic acid so-lutions of known concentrations. For folate composition, larvae were trypsinized to collect the dispersed embryonic cells. After homogenized, heated and centrifuged, the supernatants were harvested and incubated with folate enzymes and subjected to an Aquasil C18 column, 150 × 4.6 mm, 3μm (Thermo Electron Corporation, USA) on an HPLC
Fig. 2. The impact of folate supplementation on the UVB-inflicted damages in heat-shocked wild-type larvae. (A–D) Representative images of the scoring criteria for larvalfin damage at 1 dpuv. Images shown are the lateral view of whole larva (left panel) and the magnification of the tail region (right panel). Larvae were scored zero (A; intact), one (B; mild shrinkage), two (C; severe shrinkage) or three (D; disrupted) based on the extent of tailfin damages. (E) Representative images of ROS staining in larvae without UVB irradiation (WT), and in UVB-irradiated larvae (WT UVB) with/without 20μM of NAC supplementation. Larvae displaying sig-nificantly increased ROS are indicated by arrows. (F–G) The impact of supplementation with 20 μM NAC (F) and 500 μM folate adducts (G) on the extent of UVB-inflicted damage was quantified based on the score of fin morphology as illustrated in (A–D). Presented are the average of at least four independent experiments with total of 25–79 larvae for each group. WT, wild-type; FA, folic acid; 5-CHO, 5-formyltetrahydrofolate; NAC, N-acetyl-L-cysteine. The symbols “#” and “*” represent the statistical significance for the improved and worsened phenotypes, respectively. ###, ***, p < 0.001.
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system (Agilent 1100) equipped with fluorescence detector (λex = 290 nm and λem = 360 nm) for folate analysis. Solvent A was 30 mM phosphoric acid, pH 2.3, and solvent B was acetonitrile. Briefly, the column was equilibrated with 6% solvent B (94% solvent A). After sample injection, the 6% solvent B was maintained for 5 min and then over the next 20 min solvent B was linearly increased to 25% and held at this level for an additional 2 min. Then the solvent composition was decreased to 6% solvent B in 1 min and the column equilibrated for an additional 20 min before the next sample injection. Theflow rate was 0.4 ml/min. The potential folate peaks in extracts were identified by overlapping the retention times between the prospective folate peaks and folate standards.
2.9. Statistical analysis
All the data applied to the statistical analysis in this study was
collected from at least three independent experiments. One-tailed Mann-Whitney nonparametric U test was performed to calculate the probability value (P value) at 95% confidence intervals using the soft-ware GraphPad Prism 5.
3. Results
3.1. Folate supplementation ameliorated the UVB-induced injury in wild-type larvae
Our results showed that UVB irradiation caused significant mor-phological abnormalities in wild-type larvae. UVB-exposed larvae dis-played apparent shrunkenfins, especially in the tail region, at 1-day-post-UVB exposure (dpuv) (Fig. 2A–C). Mortality and disrupted tail fin occurred occasionally in those larvae with more severe damage (Fig. 2D). Increased H2DCFDAfluorescent signal was observed in
UVB-Fig. 3. The UVB-inflicted damages observed in folate-deficient larvae. Larvae with/without FD were imaged for fluorescence intensity (A) and measured for total intracellular folate content (B) at 2- and 6-dpf. (C) Larval mortality after UVB exposure was recorded at 7-dpf (1-dpuv). (D–F) Representative images of UVB-irradiated larvae with/without FD in lateral view (left panel) and the magnification of the tail region (right panel). (G) The impact of UVB-inflicted tail fin damages in larvae with various degree of FD was measured. Significantly increased tail fin damage was found in FD larvae exposed to UVB radiation. The reported data were the averages of at least three independent experiments with the total of 51–152 larvae for each group. WT, wild-type; CTL, heat-shocked non-fluorescent transgenic control; MFD, mild folate deficiency; SFD, severe folate deficiency; FD, folate deficiency. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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exposed larvae, suggesting an elevated ROS level (Fig. 2E). NAC is a reducing agent commonly used in laboratory for providing antioxidant activity. Supplementing with NAC significantly ameliorated the in-creased ROS level and tailfin damage in UVB-exposed group (Fig. 2E and F). 5-CHO-THF is the reduced folate often used clinically for
rescuing folate deficiency in patient receiving anti-folate chemotherapy (Fig. 1D). Folate supplementation, either folic acid or 5-CHO-THF, also successfully improved the UVB-induced damage (Fig. 2G). These data demonstrate the protective effect provided by folate for relieving UV-inflicted damage and support the use of zebrafish in studying the role of
Fig. 4. The antioxidant capacity and oxidative stress in folate-deficient larvae with/without UVB-irradiation. (A–B) Larvae in both control and FD groups were
Fig. 4. The antioxidant capacity and oxidative stress in folate-deficient larvae with/without UVB-irradiation. (A–B) Larvae in both control and FD groups were