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DOI 10.1002/mnfr.201300404

RESEARCH ARTICLE

Selenite protects Caenorhabditis elegans from oxidative stress via DAF-16 and TRXR-1 Wen-Hsuan Li, Yeu-Ching Shi, Chun-Han Chang, Chi-Wei Huang and Vivian Hsiu-Chuan Liao Department of Bioenvironmental Systems Engineering, National Taiwan University, Taipei, Taiwan Scope: Selenium is an essential micronutrient. In the present study, trace amount of selenite (0.01 ␮M) was evaluated for oxidative stress resistance and potential associated factors in Caenorhabditis elegans. Methods and results: Selenite-treated C. elegans showed an increased survival under oxidative stress and thermal stress compared to untreated controls. Further studies demonstrated that the significant stress resistance of selenite on C. elegans could be attributed to its in vivo free radicalscavenging ability. We also found that the oxidative and thermal stress resistance phenotypes by selenite were absent from the forkhead transcription factor daf-16 mutant worms. Moreover, selenite influenced the subcellular distribution of DAF-16 in C. elegans. Furthermore, selenite increased mRNA levels of stress-resistance-related proteins, including superoxide dismutase3 and heat shock protein-16.2. Additionally, selenite (0.01 ␮M) upregulated expressions of transgenic C. elegans carrying sod-3::green fluorescent protein (GFP) and hsp-16.2::GFP, whereas this effect was abolished by feeding daf-16 RNA interference in C. elegans. Finally, unlike the wild-type N2 worms, the oxidative stress resistance phenotypes by selenite were both absent from the C. elegans selenoprotein trxr-1 mutant worms and trxr-1 mutants feeding with daf-16 RNA interference. Conclusion: These findings suggest that the antioxidant effects of selenite in C. elegans are mediated via DAF-16 and TRXR-1.

Received: June 3, 2013 Revised: August 20, 2013 Accepted: September 5, 2013

Keywords: Caenorhabditis elegans / DAF-16 / Oxidative stress / Selenium / TRXR-1

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Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

Selenium is an essential trace nutrient that has a narrow exposure window between its beneficial and detrimental effects. The toxicity of selenium depends not only on the selenium compound and dose but also on the method of administration, exposure time, animal species, physiological status, and interaction with other metals, nutrients, etc. [1]. Industrial Correspondence: Professor Vivian Hsiu-Chuan Liao, Department of Bioenvironmental Systems Engineering, National Taiwan University, No. 1 Roosevelt Road, Sec. 4, Taipei 106, Taiwan Fax: +886-2-33663462 E-mail: [email protected] Abbreviations: DCF, dichlorofluorescein; FOXO, forkhead; GFP, green fluorescent protein; HSP, heat shock protein; RNAi, RNA interference; ROS, reactive oxygen species; SOD, superoxide dismutase  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

and agricultural activities have accelerated the release of selenium to the environment-affecting organisms in aquatic and terrestrial ecosystems around the globe [2]. Selenium is fundamentally important to human health. It is essential for the normal functioning of major metabolic pathways, including thyroid hormone metabolism, antioxidant defense systems, immune function, making selenium an essential element for normal development, growth, metabolism, and defense of the body [3, 4]. Despite its protective functions, excess amounts of selenium can be genotoxic and possibly carcinogenic [5]. In most animals, selenium is an integral part of more than about 30 known proteins. Proteins that require selenocysteine for their function are referred to as selenoproteins, and include glutathione peroxidases (GPx), thioredoxin reductases (TrxR), iodothyronine deiodinases, selenoprotein P (SeP), selenoprotein W (SeW), and selenophosphate synthetase [6–10]. Selenium functions as a redox gatekeeper through its incorporation into www.mnf-journal.com

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selenoproteins, such as GPXs and TrxRs, to alleviate oxidative stress in cells [11, 12]. The mammalian selenoprotein TrxRs are seleniumcontaining flavoprotein oxidoreductases, dependent upon a selenocysteine residue for reduction of the active site disulfide in thioredoxins [13]. TrxRs are targeted by a number of drugs having effects on cell function and promoting oxidative stress, some of which are used to treat cancer or other diseases [13]. However, potential specific or essential roles for different forms of mammalian TrxRs in health or disease are still rather unclear. In contrast to other animals, TRXR-1, an ortholog of the human enzymatic antioxidant thioredoxin reductase-1, has been suggested to be the only selenoprotein in Caenorhabditis elegans [14–16]. Unlike mammalian TrxRs, recent studies showed that C. elegans trxr-1 null mutant did not show increased sensitivity to oxidative stress after incubation with H2 O2 or paraquat [17, 18]. Therefore, the regulation and function of thioredoxin reductases within the cellular context and in intact animals require further elucidated. In C. elegans, DAF-16/forkhead (FOXO) transcription factor is an important regulator of oxidative stress resistance [19, 20]. Besides antioxidative defense, the C. elegans DAF-16 functions as a central regulator of multiple biological processes, such as longevity, fat storage, stress response, development, and reproduction [19–21]. The C. elegans DAF-16 is the human homolog yet it is the only FOXO protein in C. elegans [22]. In mammals, four FOXO genes have been identified: FOXO1, FOXO3, FOXO4, and FOXO6 [23]. Target genes of FOXOs involved in conferring stress resistance in response to FOXOs activation include those of regulators of cell-cycle progression [24], proteins associated with DNA repair [25], or the antioxidant enzymes, manganese-superoxide dismutase (MnSOD) [26] and catalase [27]. We previously showed that selenite exerts both ameliorative and toxic effects on C. elegans, depending on the amount [28]. To further explore our understanding in the beneficial effects by selenite in C. elegans, herein selenite was evaluated for oxidative stress resistance and the potential associated regulatory factors in C. elegans. Our findings demonstrated that the antioxidant effects of trace amount of selenite (0.01 ␮M) in C. elegans are mediated via DAF-16 and TRXR-1.

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by the NIH National Center for Research Resources. Mutants (trxr-1(sv43) III and trxr-1(sv47) III) were obtained from Dr. Simon Tuck’s lab (Umea˚ University, Umea, ˚ Sweden). Worms were maintained and assayed (unless otherwise stated) at 20⬚C on nematode growth medium agar plates carrying a lawn of E. coli OP50 bacteria [31]. Synchronization of worm cultures was achieved by hypochlorite treatment of gravid hermaphrodites [32].

2.2 Stress resistance assays and RNA interference Synchronized L1 larvae (wild-type or mutants) were incubated in liquid S-basal containing E. coli OP50 bacteria at 109 cells/mL and 0.01 ␮M selenite for 72 h. Subsequently, adult worms were immediately subjected to oxidative stress and heat shock assays. For the oxidative stress assay, juglone (5-hydroxy-1, 4-naphthoquinone) (Sigma-Aldrich), paraquat (1, 1 -dimethyl-4,4 -bipyridinium dichloride) (Sigma-Aldrich), and H2 O2 (Sigma-Aldrich) were used to induce oxidative stress in worms. Selenite-treated and control adult worms were exposed to 250 ␮M of juglone for 2.5 h [33], 200 mM paraquat for 3 h [34], and 2 mM H2 O2 for 3 h [17], respectively, and then scored for viability. To assess thermal tolerance, selenite-treated and control adult worms were transferred to nematode growth medium plates at 35⬚C for 7 h and then scored for viability. The survival of worms was determined by touch-provoked movement [35]. Worms were scored as dead when they failed to respond to repeated touching with a platinum wire pick. The tests were performed at least three times. RNA interference (RNAi) experiments were performed by feeding nematodes with bacteria that expressed daf-16 doublestranded (ds)RNA [36]. RNAi experiments were performed as previously described [36, 37]. Synchronized L1-stage larvae (wild-type or trxr-1 mutants) were cultured in liquid S-basal containing E. coli HT115 (DE3) bacteria with either the daf-16 RNAi clone or an empty RNAi vector (pPD129.36) [36] in the absence or presence of 0.01 ␮M selenite for 72 h at 20⬚C. Subsequently, the adult worms were immediately tested by oxidative stress challenge and then scored for viability. The tests were performed at least three times.

Materials and methods

2.1 Chemicals, C. elegans strains, and growth conditions Inorganic selenite (Na2 SeO3 ) was purchased from SigmaAldrich (Poole, Dorset, UK). Strains used in this study were Bristol N2 (wild-type); GR1307, daf-16 (mgDf50); TK22, mev1(kn1); CL2070, dvIs70[hsp-16.2:: green fluorescent protein (GFP)] [29]; CF1553, muIs84[pAD76(sod-3::GFP)] [30]; TJ356, zIs356[DAF-16::GFP]; trxr-1(sv43) III; trxr-1(sv47) III. All nematodes strains except for trxr-1 used in this work were provided by the Caenorhabditis Genetics Center, which is funded  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.3 Measurement of intracellular reactive oxygen species Wild-type N2 and mev-1 mutant worms were raised from L1 larvae as described in the stress resistance assays. Subsequently, intracellular reactive oxygen species (ROS) in C. elegans were measured using 2 ,7 -dichlorodihydrofluoroscein diacetate (H2 DCFDA) (Sigma-Aldrich). Approximately 100 nematodes were sonicated after selenite treatment, and then the worm lysates were collected for the ROS measurement [38, 39]. Worm samples were incubated with H2 DCFDA (at a final concentration of 50 ␮M in PBS) in an FLx800 Microplate www.mnf-journal.com

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Fluorescent Reader (Bio-Tek Instruments, Winookski, VT, USA) for quantification of fluorescence with excitation at 485 nm and emission at 530 nm. Samples were read for 2.5 h.

2.4 DAF-16 localization assays Synchronized L1 larvae of the TJ356 transgenic strain stably expressing a DAF-16::GFP fusion protein as a reporter [22] were incubated in liquid S-basal containing E. coli OP50 bacteria at 109 cells/mL and a final concentration of 0.01 ␮M selenite for 72 h at 20⬚C. Subsequently, adult TJ356 transgenic strain was treated with 50 ␮M juglone for 5 min. Subsequent to this treatment, worms were placed on microscope slides and capped with coverslips, and the subcellular DAF-16 distribution was analyzed by fluorescence microscopy on an epifluorescence microscope (Leica, Wetzlar, Germany) using a suited filter set (excitation at 480 ± 20 nm; emission at 510 ± 20 nm) with a cooled charge coupled device camera. Expression patterns of TJ356 worms were classified into three categories (cytosolic, intermediate, and nuclear) with respect to major localization of the DAF-16::GFP fusion protein. The images were photographed and the fluorescence intensities were directly analyzed using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA).

2.5 Quantitative real-time RT-PCR analysis Synchronized L1 wild-type larvae were incubated in liquid S-basal containing E. coli OP50 bacteria at 109 cells/mL and 0.01 ␮M selenite for 72 h at 20⬚C. Subsequently, adult worms were challenged by 150 ␮M juglone at 20⬚C for 1 h to generate oxidative stress to induce gene expression. After stress induction, worms were immediately washed three times by S-basal medium. Total RNAs were extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Total RNA (2 ␮g) was reverse transcribed using the reverse transcriptase enzyme Superscript III (Invitrogen) and an oligo-dT primer (Sigma-Aldrich). Real-time PCR was performed with Power SYBR Green PCR Master Mix (Qiagen, Hilden, Germany) using Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primer sequence is presented in Supporting Information Table 1. The relative quantities of mRNA were determined using comparative cycle threshold methods and normalized against the myosin light chain gene (mlc-2) mRNA [40–42]. The test was performed three times.

2.6 Induction of stress response reporter and RNAi RNAi experiments were performed essentially as described in stress resistance assays. Synchronized L1-stage transgenic larvae with inducible GFP reporter for hsp-16.2 (CL2070  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

strain) and sod-3 (CF1553 strain) were cultured in liquid S-basal containing E. coli HT115 (DE3) bacteria with either the daf-16 RNAi clone or an empty RNAi vector (pPD129.36) [36] in the absence or presence of 0.01 ␮M selenite for 72 h at 20⬚C. Subsequently, worms with GFP reporter for hsp-16.2 were heat-shocked at 35⬚C for 2 h and recovered at 20⬚C for 4 h [43], whereas sod-3::GFP worms were incubated for 1 h at 20⬚C in liquid medium containing 150 ␮M juglone to generate oxidative stress [44]. The expressions of hsp-16.2 and sod-3 were directly measured by observing the fluorescence of the reporter GFP. To analyze GFP fluorescence, 30 randomly selected worms from each set of experiments were mounted onto microscope slides coated with 2% agarose, anesthetized with 2% sodium azide, and capped with coverslips. Epifluorescence images were captured with an epifluorescence microscope (Leica) using a suited filter set (excitation at 480 ± 20 nm; emission at 510 ± 20 nm) with a cooled charge coupled device camera. Adult worms were examined and total GFP fluorescence for each whole worm was quantified by ImagePro Plus software (Media Cybernetics).

2.7 Data analysis Statistical analysis was performed using SPSS, version 13.0 (SPSS Inc., Chicago, IL, USA). Results are presented as the mean ± SEM. The statistical significance of differences between the populations was demonstrated by one-way ANOVA and LSD post hoc test. Differences were considered significant at p < 0.05 (* ), p < 0.01 (** ), or p < 0.001 (*** ) (see figures).

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Results

3.1 Selenite improves the stress resistance of wild-type C. elegans We previously showed that 0.01 ␮M selenite exerts ameliorative effects on development, reproduction, and cholinergic signaling in C. elegans [28]. In the present study, we therefore selected 0.01 ␮M selenite as the working concentration to explore selenite’s protective actions in C. elegans. To investigate whether selenite has protective effects in C. elegans under oxidative and thermal stress, wild-type N2 worms were pretreated with selenite followed by exposure to oxidative and thermal stresses. To evaluate the potential effect of selenite on C. elegans under oxidative stress, wild-type N2 synchronized L1 larvae were pretreated with 0.01 ␮M selenite for 72 h at 20⬚C before being exposed to juglone (250 ␮M), a redox cycler that generates intracellular oxidative stress [45], and then incubated for 2.5 h at 20⬚C. During pretreatment with 0.01 ␮M selenite, no adverse effects on the worms, including survival, the growth rate, progeny www.mnf-journal.com

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Worms were then subjected to heat shock treatment by placing them onto nematode growth medium plates at 35⬚C for 7 h, and viability was scored. Results showed that 0.01 ␮M selenite pretreatment enhanced the worms’ resistance to thermal stress, thus generating a 30% increase in the survival of worms under heat shock treatment (Fig. 1). Taken together, trace amount of selenite (0.01 ␮M) could enhance the stress-resistance of C. elegans under oxidative and thermal stress, suggesting that selenite might provide these protective effects by lowering intracellular free radicals level as the toxicity of oxidative stress and thermal stress associated with damage caused by accumulation of ROS has been described [47–49]. Figure 1. Protective effects of selenite on wild-type Caenorhabditis elegans N2 under oxidative stress and thermal stress. Synchronized wild-type L1 larvae were pretreated with selenite (0.01 ␮M) or distilled water as control (0 ␮M) for 72 h at 20⬚C. Subsequently, adult worms were immediately subjected to oxidative stress and heat shock assays. For the oxidative stress assays, selenite-treated (0.01 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) and control (0 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) adult worms were exposed to 250 ␮M juglone for 2.5 h, 200 mM paraquat for 3 h, and 2 mM H2 O2 for 3 h, respectively, at 20⬚C and then scored for viability. To assess the thermal tolerance, selenite-treated (0.01 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) and control (0 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) adult worms were incubated at 35⬚C for 7 h and then scored for viability. Results are presented as the mean ± SEM. Differences compared to the control (0 ␮M) were considered significant at p < 0.01 (**) and p < 0.001 (***) by one-way ANOVA and LSD post hoc test.

production, body length, or morphological changes, were observed. The results showed that 0.01 ␮M selenite pretreatment resulted in a significant increase in the survival for worms exposed to juglone-induced oxidative stress (Fig. 1). The survival of control worms to juglone exposure (Fig. 1) showed slightly different sensitivity from other reports [33, 46]. This might be due to different developmental stages and exposure scenarios/conditions. In addition to juglone, we further used two additional oxidative stress generators (200 mM paraquat and 2 mM H2 O2 ) to examine the protective potential of selenite on C. elegans against oxidative stress. The results showed that 0.01 ␮M selenite pretreatment also significantly increased the survival of worms under paraquat and H2 O2 challenges (Fig. 1). Together, the results demonstrated that selenite (0.01 ␮M) increases the resistance to oxidative stress generated by different oxidative stress generators in C. elegans, suggesting that antioxidative potential of trace amount of selenite is not limited to the form of ROS. To assess thermal stress, wild-type N2 synchronized L1 larvae were pretreated with 0.01 ␮M selenite for 72 h at 20⬚C.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.2 Selenite decreases the intracellular ROS level in C. elegans The free radical scavenging abilities of selenite in C. elegans were evaluated. Wild-type animals were raised from L1 larvae as described in the stress resistance assays. Subsequently, intracellular ROS for adult worms was measured using 2’,7’dichlorodihydrofluoroscein diacetate (H2 DCF-DA) [38, 39]. Nonfluorescent DCF-DA is a freely cell permeable dye, which is readily converted to fluorescent 2’7’-dichlorofluorescein (DCF) due to the interaction with intracellular peroxide (H2 O2 ). The result showed that selenite at the concentration of 0.01 ␮M could significantly inhibit the production of ROS in vivo (compared with the control, p < 0.001) (Fig. 2A). The free radical scavenging abilities of selenite were further evaluated by using the oxidative stress-hypersensitive mev-1 mutant. MEV-1 is a subunit of succinate-coenzyme Q oxidoreductase in complex II of the electron transport chain [50], and the mev-1 mutant is hypersensitive to oxidative stress, mostly due to mev-1 mitochondrial ROS overproduction. Synchronized L1 mev-1 mutant larvae were pretreated with 0.01 ␮M selenite for 72 h at 20⬚C before being exposed to juglone (250 ␮M) and then incubated for 2.5 h at 20⬚C and then the survival of worms was scored. Additionally, before being exposed to juglone, mev-1 mutant worms were directly prepared for intracellular ROS measurement. Figure 2A showed that the ROS level in mev-1 mutant worms was significantly higher than that wild-type N2 worms without selenite supplementation (p < 0.001). In addition, selenite (0.01 ␮M) significantly attenuated the ROS overproduction in mev-1 mutant worms (compared to the untreated control of mev-1 mutant strain, p < 0.001, Fig. 2A). Moreover, the results showed that supplementation of 0.01 ␮M selenite significantly enhanced the survival of mev-1 mutant against juglone exposure and heat shock treatment, respectively, compared with the untreated ones (p < 0.001, Fig. 2B). Taken together, supplementation of 0.01 ␮M selenite could enhance the survival of C. elegans by alleviating the accumulation of intracellular ROS level during normal culture conditions and environmental stress. www.mnf-journal.com

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Figure 3. Effects of selenite on daf-16 mutant under oxidative stress and thermal stress. Synchronized daf-16 mutant L1 larvae were pretreated with selenite (0.01 ␮M) or distilled water as control (0 ␮M) for 72 h at 20⬚C. Subsequently, adult worms were immediately subjected to juglone-induced oxidative stress assays. Selenite-treated (0.01 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) and control (0 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) adult worms were subjected to stress challenges as described in the wild-type stress resistance assays and then scored for viability. Results are presented as the mean ± SEM. Differences compared to the control (0 ␮M) were considered significant at p < 0.05 by one-way ANOVA and LSD post hoc test. ns, not significant.

3.3 Selenite enhances oxidative stress resistance and thermal tolerance in C. elegans via DAF-16

Figure 2. Free radical-scavenging effects of selenite in vivo. (A) Synchronized wild-type N2 and mev-1 mutant L1 larvae were pretreated with selenite (0.01 ␮M, n = 3–4 independent experiments, approximately 100 worms were assayed in each experiment) or distilled water as control (0 ␮M, n = 3–4 independent experiments, approximately 100 worms were assayed in each experiment) for 72 h at 20⬚C. Subsequently, intracellular ROS for adult worms was measured by using 2’, 7’-dichlorodihydrofluoroscein diacetate. Results are expressed as relative fluorescence units to each worm. (B) Synchronized TK22 (mev-1) mutant worms were raised from L1 larvae and pretreated with selenite (0.01 ␮M) or distilled water as control (0 ␮M) for 72 h at 20⬚C. For the oxidative stress assays, selenite-treated (0.01 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) and control (0 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) worms were exposed to 250 ␮M of juglone for 2.5 h at 20⬚C and then scored for viability. To assess the thermal tolerance, selenite-treated (0.01 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) and control (0 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) adult worms were incubated at 35⬚C for 7 h and then scored for viability. Results are presented as the mean ± SEM. Differences compared to the control (0 ␮M) were considered significant at p < 0.001 (***) by one-way ANOVA and LSD post hoc test.

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In C. elegans, the DAF-16/FOXO signaling pathway functions to modulate longevity, stress resistance, and reproductive development [19]. To investigate whether selenite-enhanced stress resistances were mediated by DAF-16/FOXO, we examined worms that lack of daf-16 gene in response to selenite. Unlike the wild-type N2 worms, after 0.01 ␮M selenite treatment for 3 days at 20⬚C followed by juglone exposure and thermal challenge, daf-16 mutant worms did not show significantly increased survival compared to that untreated ones (Fig. 3). This suggests that the selenite may provide oxidative stress and thermal resistance in C. elegans via DAF-16.

3.4 Selenite induces the translocation of DAF-16 from cytoplasm to nucleus To further explore the role of DAF-16 in modulating seleniteenhanced oxidative-stress resistance, we examined the nuclear translocation of DAF-16. DAF-16 is a key factor in insulin signaling pathway. DAF-16 is a FOXO transcription factor with specific functions such as upregulating antioxidant-defense and DNA repair-facilitating genes, and localization of DAF-16 in nuclei is an essential prerequisite for its ability to activate target gene transcription [51–54]. Synchronized L1 transgenic TJ356 strain (DAF-16::GFP) was incubated with 0.01 ␮M selenite in liquid S-basal for 72 h at 20⬚C www.mnf-journal.com

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Figure 4. Influences of selenite on subcellular DAF-16 localization under oxidative stress induction. Synchronized L1 transgenic TJ356 strain (DAF16::GFP) were pretreated with 0.01 ␮M selenite or distilled water as control (0 ␮M) in liquid S-basal for 72 h at 20⬚C to reach adulthood. Subsequently, the TJ356 strain was challenged with 50 ␮M juglone for 5 min, and then the fluorescence intensity was measured. (A) The worms were classified to the categories “cytosolic” (left panel), “intermediate” (middle panel), and “nuclear” (right panel) according to their localization phenotypes. (B) Comparative evaluation of subcellular distribution of DAF-16 in each group. Percentage was calculated by the presence of worms in the category normalizing to the total population of worms in each treatment condition (n = 30 for each condition). Results are presented as the mean ± SEM. Differences compared to the control (0 ␮M) were considered significant at p < 0.001 (***) by one-way ANOVA and LSD post hoc test.

to reach adulthood. Subsequently, the TJ356 strain was challenged with 50 ␮M juglone for 5 min, and then the fluorescence intensity was measured. Figure 4A shows the representative images of localization phenotypes of TJ356 worms. The results showed that transgenic TJ356 strain (DAF-16::GFP) without selenite treatment revealed a predominant cytosolic localization of DAF-16 (Fig. 4B). In contrast, treatment of TJ356 strain with 0.01 ␮M selenite resulted in a significant decrease in the fractions of worms with cytosolic phenotypes (p < 0.001), whereas significant increases in the fractions of worms with intermediate (p < 0.001) and nuclear (p < 0.001) localization phenotypes (Fig. 4B). Therefore, selenite affected the subcellular distribution of DAF-16 and caused translocation of DAF-16 from the cytoplasm to nuclei.

3.5 Selenite enhances expressions of small HSP-16.2 and SOD-3 in C. elegans via DAF-16 To elucidate whether the above-described increase in stress resistance was due to selenite regulating DAF-16-dependent stress-response gene expressions, we examined the respon C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

siveness of heat shock protein (HSP) and the antioxidant enzyme superoxide dismutase (SOD) to selenite treatment. Under physiological conditions, small heat shock proteins (sHSPs) are among the most highly inducible HSPs during thermal and oxidative stress. The HSP-16.2 family of proteins is homologous to ␣B crystalline and expressed under conditions of stress in C. elegans [43]. SOD is a major enzyme that protects against oxidative stress by catalyzing the removal of O2 − [55]. The C. elegans manganese superoxide dismutase SOD-3 is an antioxidative enzyme that is induced in response to stress [56, 57]. Both hsp-16.2 and sod-3 have been implicated as target genes of DAF-16 [58]. We examined the changes of mRNA levels of SOD-3 and HSP-16.2 in selenite treated and control worms by real-time RT–PCR assays. The results showed that supplementation of selenite (0.01 ␮M) significantly increased mRNA levels of SOD-3 and HSP-16.2, upon oxidative stress induction, compared to the untreated group (Fig. 5A). Furthermore, we treated the transgenic C. elegans (CL2070 and CF1553) expressing GFP as a reporter transgene for inducible hsp-16.2 and sod-3 expression, respectively, with selenite (0.01 ␮M). The CL2070 strain followed by heat shock www.mnf-journal.com

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7 at 35⬚C for 2 h and recovery at 20⬚C for 4 h, and then the fluorescence intensity was measured. Figure 5B showed that the expression of hsp-16.2 induced by heat shock was significantly enhanced in CL2070 worms fed with 0.01 ␮M selenite compared to that untreated ones (p < 0.001). The CF1553 strain was challenged by 150 ␮M juglone for 1 h at 20⬚C, and then the fluorescence intensity was measured. The results showed that the expression level of sod-3 was significantly increased in CF1553 worms treated with 0.01 ␮M selenite compared to that untreated ones (p < 0.001, Fig. 5B). To further confirm that selenite-enhanced expressions of hsp-16.2 and sod-3 were mediated by DAF-16, we performed RNAi experiments by feeding CL2070 and CF1553, respectively, with E. coli that had been transformed with a plasmid that expresses daf-16 dsRNA. Expressions of hsp-16.2 and sod-3 were decreased by feeding worms with daf-16 dsRNA (Fig. 5B). In addition, 0.01 ␮M selenite-enhanced expressions of hsp-16.2 and sod-3 were abolished (compared to worms with 0 ␮M selenite and daf-16 RNAi treatment, Fig. 5B). Taken together, the results indicate that DAF-16 is required for selenite-enhanced expressions of small HSP-16.2 and SOD-3 in C. elegans.

3.6 Both TRXR-1 and DAF-16 contribute to selenite-enhanced oxidative stress resistance in C. elegans

Figure 5. Effects of selenite on antioxdative genes (hsp-16.2 and sod-3) expression regulated by DAF-16. (A) Synchronized wildtype L1 larvae were incubated with 0.01 ␮M selenite or distilled water as control (0 ␮M) in liquid S-basal for 72 h at 20⬚C. The adult worms were then challenged with 150 ␮M juglone for 1 h. Total RNA was extracted and mRNA levels of HSP-16.2 and SOD3 were determined by quantitative real-time PCR (qRT-PCR). All measurements were normalized to mRNA level of MLC-2 (myosin light chain), and fold change of each gene was normalized to that observed in the control (0 ␮M) samples. (B) Immediately after hatching, synchronized L1 transgenic CL2070 strain (hsp16.2::GFP) and CF1553 strain (sod-3::GFP) were incubated with 0.01 ␮M selenite or distilled water as control (0 ␮M) in liquid S-basal for 72 h at 20⬚C. During the incubation, worms were fed with E. coli OP50 or HT115 (DE3) bacteria containing the daf-16 RNAi clone or an empty RNAi vector (pPD129.36) as food source. The CL2070 strain followed by heat shock at 35⬚C for 2 h and recovery at 20⬚C for 4 h, and then the fluorescence intensity was measured. The CF1553 strain was challenged by 150 ␮M juglone for 1 h at 20⬚C, and then the fluorescence intensity was measured. Total GFP fluorescence intensity for each whole worm was quantified by Image-Pro Plus software. GFP expression ratio was calculated by the average GFP intensity in each treatment condition (n = 30 for each condition) normalizing to that of control (0 ␮M selenite, n = 30). Results are presented as the mean ± SEM. Differences compared to the control (0 ␮M) were considered significant at p < 0.01 (**) and p < 0.001 (***) by one-way ANOVA and LSD post hoc test. ns, not significant.

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To further explore factors might be involved in modulating selenite-enhanced oxidative-stress resistance in C. elegans, we examined the roles of the sole C. elegans selenoprotein TRXR1 in response to selenite-enhanced oxidative-stress resistance. We firstly investigated whether TRXR-1 is involved in oxidative stress in C. elegans. We examined worms that lack of trxr-1 gene in response to oxidative stress. The results showed that both trxr-1(sv43) and trxr-1(sv47) mutant worms did not show significantly different survival from that wild-type N2 after juglone and paraquat exposure, respectively (Fig. 6A). This suggests that TRXR-1 might not be directly involved in oxidative stress regulation in C. elegans. We next examined whether TRXR-1 plays a role in seleniteenhanced oxidative stress resistances. trxr-1 mutant worms were exposed to selenite followed by oxidative stress challenge. Unlike the wild-type N2 worms, after 0.01 ␮M selenite treatment for 3 days at 20⬚C followed by juglone exposure, both trxr-1(sv43) and trxr-1(sv47) mutant worms did not show significantly increased survival compared to that untreated ones (Fig. 6B). This suggests that the selenite may provide oxidative stress resistance in C. elegans via TRXR-1. We further investigated the relationship of DAF-16 and TRXR-1 in mediating selenite-enhanced oxidative stress resistance in C. elegans. We performed RNAi experiments by feeding trxr-1(sv43) and trxr-1(sv47) mutant worms, respectively, with E. coli that had been transformed with a plasmid that expresses daf-16 dsRNA followed by juglone exposure. In the absence of selenite, the results showed that the daf-16 www.mnf-journal.com

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RNAi partly suppressed the survival phenotype of trxr-1(sv43) and trxr-1(sv47) mutants (Fig. 6B). Yet the survival phenotypes of trxr-1(sv43);daf-16 RNAi and trxr-1(sv47);daf-16 RNAi is still slightly higher than that of daf-16 RNAi alone (Fig. 6B). Similarly, in the presence of selenite, the survival phenotypes of trxr-1(sv43);daf-16 RNAi and trxr-1(sv47);daf-16 RNAi are higher than that of daf-16 RNAi and lower than those of trxr1(sv43) and trxr-1(sv47) mutants (Fig. 6B). Taken together, the results suggest that both DAF-16 and TRXR-1 contributed to selenite-enhanced oxidative stress resistance in C. elegans.

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Figure 6. Effects of selenite on trxr-1 under oxidative stress. (A) Synchronized adult worms (wild-type N2, trxr-1(sv43), and trxr1(sv47); n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) were exposed to 250 ␮M of juglone for 2.5 h and 200 mM paraquat for 3 h, respectively, at 20⬚C and then scored for viability. (B) Synchronized wild-type N2 and trxr-1 mutant animals were raised from L1 larvae and pretreated with selenite (0.01 ␮M) or distilled water as control (0 ␮M) for 72 h at 20⬚C. For RNAi assays, the liquid cultures contained E. coli HT115 (DE3) bacteria with either the daf-16 RNAi clone or an empty RNAi vector (pPD129.36). Selenite-treated (0.01 ␮M, n = 3–5 independent experiments, approximately 60– 80 worms were scored in each experiment) and control (0 ␮M, n = 3–5 independent experiments, approximately 60–80 worms were scored in each experiment) adult worms were exposed to 250 ␮M of juglone for 2.5 h at 20⬚C and then scored for viability. The tests were performed at least three times. Results are presented as the mean ± SEM. Differences (selenite-treated versus control (0 ␮M)) were considered significant at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) by one-way ANOVA and LSD post hoc test. ns, not significant. Significant differences between strains (0 ␮M, lowercase letters; 0.01 ␮M selenite-treated, uppercase letters) at the p < 0.05 level were tested using one-way ANOVA and LSD post hoc test. Means with different letters were significantly different in pairwise comparisons.

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Discussion

Selenium is an essential trace nutrient that functions as antioxidant whereas at higher concentrations, selenium is prooxidant and toxic. The antioxidant and toxic properties of selenium have been intensively examined in cell culturebased mammalian systems but less result was from in vivo studies. Oxidative stress is regarded as a main factor in the pathophysiology of various diseases and ageing [59] and it occurs as a result of excessive generation of ROS or diminished antioxidative defense systems. To regulate the overall ROS levels generated from endogenous and/or exogenous sources and protect the cells from stress condition, antioxidant defenses systems and mechanisms are necessary [49]. Morgan et al. showed that selenite both prevents and induces oxidative stress through a process that involves the GLRX-21 glutaredoxin in C. elegans [60]. Yet, the mechanism through which selenite operates the antioxidant property is not fully understood. In our previous study, selenite has been shown to have the ameliorative and toxic effects on development, reproduction, and cholinergic signaling in C. elegans [28]. In this study, we observed that trace amount of selenite (0.01 ␮M) could significantly enhance the survival of C. elegans under heat stress and oxidative stress (Fig. 1). This demonstrates that selenite has antioxidant-like properties in C. elegans. An important feature of the toxicity of oxidative stress is presumably the elevated generation of ROS [47–49]. Removal of ROS from cells occurs through the activities of one of the antioxidant enzymes such as GPx, catalase, peroxiredoxins, SOD, or by a TrxRdependent pathway [55,61,62]. We examined the influence of selenite on the intracellular amount of ROS and showed that wild-type C. elegans grown with selenite (0.01 ␮M) supplementation contained decreased levels of ROS in comparison to worms raised on a normal diet (Fig. 2A). Moreover, supplementation of selenite (0.01 ␮M) increased mRNA levels of SOD-3 (Fig. 5A) and enhanced the expression level of sod-3 in transgenic C. elegans carrying sod-3::GFP (Fig. 5B). This suggests that selenite enhanced SOD-3 activity, thereby removal of intracellular ROS in C. elegans. Additionally, TK22 (mev1), a strain known to overproduce mitochondrial ROS [50], showed an increase survival when supplemented with selenite (0.01 ␮M) (Fig. 2B). This demonstrates that selenite may www.mnf-journal.com

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against stress from ROS scavenging ability and mitochondrial ROS toxicity decline. Only a few studies investigated the roles of FOXO and selenoproteins in response to oxidative stress. One in vitro study showed stimulation of SeP promoter activity by the forkhead box transcription factor FOXO1a in hepatoma cells [63]. Yet, the mechanism through which selenite operates the antioxidant property by SeP and FOXO is not understood. In C. elegans, DAF-16/FOXO is considered a key regulator of many important biological processes including lifespan, metabolism, and oxidative stress responses [19]. We examined whether selenium homeostasis is linked to FOXOdependent signaling pathway. In contrast to the observation in wild-type C. elegans, the enhanced stress resistances against juglone-induced oxidative stress and thermal challenge by selenite were not observed in daf-16 deletion mutant (Fig. 3). The lack of stress resistance enhancement for this particular mutant suggests that the antioxidant property possessed by selenite may be dependent on DAF-16/FOXO-dependent insulin signaling pathway. To further validate that DAF-16 is required for the protective effect of the selenite, the effect of the selenite on the translocation of DAF-16 from the cytoplasm to nuclei was examined (Fig. 4). In addition, the gene expressions of the downstream effectors of DAF-16, HSP-16.2 and SOD-3, were examined after selenite treatment. Results showed that selenite was able to enhance DAF-16 translocation from the cytoplasm to nuclei under juglone-induced oxidative stress (Fig. 4). This suggests that the antioxidant property possessed by selenite may be dependent on DAF-16, thereby enhancing the expressions of DAF-16 target genes. Nuclear localization of DAF-16 is a prerequisite for transcriptional activation of its target genes such as genes for antioxidative enzymes like MnSOD (sod-3) and catalases (ctl-1 and ctl-2) [64]. We observed that 0.01 ␮M selenite caused an increase in up-regulation of hsp-16.2 and sod-3 in C. elegans upon oxidative stress (Figs. 5A, B), whereas 0.01 ␮M seleniteenhanced expressions of hsp-16.2 and sod-3 were abolished to the normal expression levels while treating with daf-16 RNAi (Fig. 5B). This indicates that DAF-16 is required for seleniteenhanced expressions of hsp-16.2 and sod-3. In addition, it might also explain why selenite could significantly increase the survival of C. elegans under thermal stress and oxidative stress. The mammalian selenoproteins, in particular TrxRs, have been implicated in countering oxidative damage and many biological processes but the molecular functions of these proteins have not been extensively investigated in different animal models [11–13, 65, 66]. Moreover, potential specific or essential roles for different forms of mammalian TrxRs in health or disease are still largely unknown. TrxRs are evolutionarily well conserved from bacteria to human. In C. elegans, the human ortholog of thioredoxin reductase-1, TRXR-1, has been suggested to be the only seleno-cysteine containing protein in C. elegans [14–16]. Recently, Stenvall et al., (2011) demonstrated that TRXR-1 does not protect against acute H2 O2 -induced oxidative stress but functions in C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

9 stead together with GSR-1 glutathione reductase to promote the removal of old cuticle during molting in C. elegans [17]. Moreover, a recent report showed that trxr-1 deletion mutant did not show hypersensitivity to paraquat exposure [18]. We examined two null trxr-1(sv43) and trxr-1(sv47) mutants [17] in response to juglone and paraquat exposure, respectively. Figure 6A showed that the results are in agreement with the previous reports [17, 18]. We further examined whether TRXR-1 plays a role in selenite-enhanced oxidative stress resistances in C. elegans. Interestingly, unlike the wild-type N2 worms, worms lacking trxr-1 did not show significantly increased survival compared to that untreated ones after 0.01 ␮M selenite pretreatment followed by juglone exposure (Fig. 6B). This suggests that TRXR-1 is involved in seleniteenhanced oxidative stress resistance in C. elegans. It has been implied that the Trx-reducing activity of mammalian TrxR is totally selenium-dependent [67]. In C. elegans, it was reported that replacement of Sec with Cys in TRXR-1 completely eliminated catalytic activity to reduce thioredoxin [17]. Therefore, this suggests that the ability of TRXR-1 to protect C. elegans from oxidative stress is selenium-dependent. Furthermore, both TRXR-1 and DAF-16 seem contributed to selenite-mediated oxidative stress resistances in C. elegans (Fig. 6B). However, whether TRXR-1 and DAF-16 act in the same or parallel pathway in response to selenite-mediated oxidative stress resistances is still unclear and required further investigation. In the present study, we investigated selenite-mediated oxidative stress resistance and the potential associated regulatory factors in C. elegans. Although most of the effects in the data are fairly moderate, the biological significance of these effects is important as many key findings with relevance for mammals were discovered in C. elegans. C. elegans is increasingly used to study the effects of pharmacologically active compounds of herbal origin on biological processes and identify new targets for pharmacological interventions [68–70]. In addition, C. elegans can provide useful information to further improve the health benefits of human beings [44,71,72]. Our study demonstrated that selenite-mediated stress resistance is modulated by DAF-16/FOXO and TRXR-1 providing new insights to understand selenium’s mode of actions in the intact organisms. The protective effects of selenite in the nematode C. elegans under stress might be attributed to its direct ROS-scavenging activities and indirect free radicalscavenging activities through upregulating target genes of DAF-16 such as hsp-16.2 and sod-3. Therefore, the protective effects of selenite is likely mediated via regulation of a FOXOs/DAF-16-dependent signaling pathway by inducing the expression of the target genes (hsp-16.2 and sod-3) of FOXOs/DAF-16 and thereby enhancing stress resistance. In addition, the sole selenoprotein in C. elegans, TRXR-1, also plays an important role in selenite-enhanced oxidative stress resistance. Moreover, both TRXR-1 and DAF-16 contribute to selenite-enhanced oxidative stress resistances These findings advance our understanding in the regulatory mechanism of selenium in the intact organisms. www.mnf-journal.com

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˚ SweWe thank Dr. Simon Tuck (Umea˚ University, Umea, den) for kindly providing trxr-1(sv43) III and trxr-1(sv47) III strains. This work was financially supported in part by grants (NSC 100-2313-B-002-013 and NSC 101-2313-B-002041-MY3) from the National Science Council of Taiwan to V. H.-C. Liao and postdoctoral fellowship grants (NSC 100-2811B-002-112 and NSC 101-2811-B-002-046) from the National Science Council of Taiwan to Y.-C. Shi. The authors have declared no conflict of interest.

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