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Feeding a ROS-generator to Caenorhabditis elegans leads to increased expression of small heat shock protein HSP-16.2 and hormesis

Feeding a ROS-generator to Caenorhabditis elegans leads to increased expression of small heat... Genes Nutr (2009) 4:59–67 DOI 10.1007/s12263-009-0113-x RESEARCH PAPER Feeding a ROS-generator to Caenorhabditis elegans leads to increased expression of small heat shock protein HSP-16.2 and hormesis Kai Hartwig Æ Tanja Heidler Æ Jan Moch Æ Hannelore Daniel Æ Uwe Wenzel Received: 16 October 2008 / Accepted: 10 February 2009 / Published online: 28 February 2009 Springer-Verlag 2009 Abstract Reactive oxygen species (ROS) are thought to Introduction be a driving force in the aging process. In transgenic Cae- norhabditis elegans expressing green fluorescent protein The exposure to high amounts of reactive oxygen species (GFP) under control of the hsp-16.2 promoter (CL2070) 100 (ROS), as occurs in many tissues, e.g. by heavy physical lM of the ROS-generator juglone induced GFP-expression. exercise, is answered by prominent adaptive responses of This was associated with translocation of DAF-16 to the the antioxidant defense system, which is of vital impor- nucleus as visualized in a transgenic strain expressing a tance in the protection against oxidative stress [13]. Small DAF-16::GFP fusion protein (TJ356) and with increased heat-shock proteins (HSPs), a family of low-molecular cellular levels of reduced glutathione. RNA-interference for weight polypeptides that are highly conserved from DAF-16 in CL2070 blocked the juglone-induced HSP-16.2 yeast to humans [6, 34], appear of general importance expression and the increase in glutathione levels. Higher for adaptations as their expression correlates usually concentrations of juglone did not further increase the well with the presence of various stressors [21, 31]. In adaptive responses but caused premature death, indicating the nematode Caenorhabditis elegans levels of small hormetic adaptations unless the stressor exceeds the intrin- HSP-16.2 on the first day of adult life predicts as much as sic protective capacity. The addition of the ROS-scavenger a fourfold variation in subsequent survival [29]. This ascorbic acid finally blocked lifespan reductions and all of correlation was highly specific for HSP-16.2 since the the adaptations to juglone stressing that ROS are indeed the induction of other genes normally activated by stress did molecular species that require protective response. not show immediate relationship to longevity [29]. Moreover, HSP-16.2 expression was identified as a good Keywords Caenorhabditis elegans  Hormesis  predictor of the ability to withstand a lethal thermal stress Reactive oxygen species  Aging  Heat shock protein  [29] and its levels correlate well with the hormetic effects Glutathione in response to heat treatment [26]. Mechanistically, HSP- 16 could affect lifespan by reducing oxidative stress through raising the pool of reduced glutathione (GSH) in the worm as suggested by Walker and Lithgow [35]. In a similar fashion HSP-27 is thought to mediate its effects in K. Hartwig  T. Heidler  H. Daniel Department of Food and Nutrition, murine cells [23, 27]. This protection against oxidative Molecular Nutrition Unit, stress appears to be accomplished by small HSPs through Technical University of Munich, a glucose-6-phosphate dehydrogenase-dependent increase Am Forum 5, 85350 Freising, Germany in NADPH generation needed to maintain GSH in its J. Moch  U. Wenzel (&) reduced form via the GSSG-reductase and by using this Molecular Nutrition Research, redox modulator as an essential cofactor of their in vivo Interdisciplinary Research Center, chaperone activity against oxidized proteins [27]. This Justus-Liebig-University of Giessen, function of HSP-16.2 in stress resistance and longevity is Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany embedded into the insulin/IGF-1 signaling pathway that e-mail: [email protected] 123 60 Genes Nutr (2009) 4:59–67 has been shown in many studies to be a central deter- Lifespan analysis minant of lifespan in C. elegans [30]. The DAF-16 transcription factor, a downstream target of the insulin/ Synchronous populations were obtained by allowing 50 IGF-1 receptor DAF-2 that translocates into the nucleus hermaphrodites to lay eggs for 3 h. Eggs were placed on 50 mm NGM plates seeded with E. coli OP50 (40–50 per in the absence of insulin/IGF-1 signaling, is essential for maximal HSP-16 expression and for lifespan extension plate, 80–300 individuals per experiment) and grown at 20C until L4 larval stage. Each population was examined conferred by it [35]. Besides the hsp-16.2 gene, however, sod-3 coding for a superoxide dismutase (SOD) and ctl-1 for synchrony at the beginning of the reproductive period. Lifespan analysis on agar plates with E. coli as the food and ctl-2 coding for catalase isoforms are further tran- scriptional targets of DAF-16 that contribute to the source was carried out according to standard protocols longevity phenotype of daf-2 mutants, probably by pre- [17]. After reaching the young adult stage worms were venting or repairing oxidative macromolecular damage, transferred to new NGM agar plates with growth arrested and knock-down of those genes by RNA-interference E. coli, which were treated before with 100 lg/ml ampi- (RNAi) reduces the lifespan in daf-2 mutants by 10–20% cillin. Worms were monitored every day and transferred to [24, 25]. fresh plates every third day. They were scored as dead when no response to a mechanical stimulus or no pharynx In the present study we assessed whether application of juglone, a generator of superoxide anion radicals [4], contraction was visible. Animals that crawled off the plates were replaced by a new worm from a substitution plate. delivered via the oral route triggers the stress response in C. elegans and in how far the adaptations to juglone enable For lifespan analysis in liquid axenic medium L4 larvae the maintenance of a normal lifespan. Nematodes were were transferred into wells containing CeHR medium and grown from the L4 stage on in an adapted liquid axenic were distributed into new wells after reaching young adult medium to assure the proper oral uptake and transport of stage. Juglone was applied in 92% ethanol, 8% Tween 80 juglone through pumping in the worm pharynx posteriorly (v/v) and diluted 1:100 in the final medium. Controls were [2]. Induction of HSP-16.2 was estimated by the expression always exposed to the same amount of solvent. Worms of green fluorescent protein (GFP) in a hsp-16.2p::gfp were moved to wells containing fresh medium and com- transgenic strain (CL2070) and translocation of DAF-16 pounds every third day with a glass capillary and scored as dead when they did not respond to a mechanical stimulus was visualized using a daf-16::gfp transgenic strain (TJ356). Activities of SOD, catalase and levels of reduced or no more pharynx contraction was seen. Experiments were started with 70 worms per group and ten worms as a (GSH) and oxidized (GSSG) glutathione were determined by UV-absorbance based assays. reserve for lost or bagged individuals. Stress resistance Methods For measuring resistance of CL2070 to heat stress nema- Nematode strains, maintenance and mass culture todes were grown in NGM medium at 20C. Therefore, approximately ten L1 larvae stage worms in 10 llM9 Nematodes were grown on NGM agar plates seeded buffer were dispensed to each well of a 96-well flat- bottomed tissue culture plate together with 40 llof with E. coli strain OP50 as described [3] and were kept at 20C. Strains used in this study were: N2, variation resuspended OP50 bacterial culture with OD = 1. After reaching the young adult stage nematodes were incubated bristol (wildtype); CL2070, dvIs70 Is[hsp-16.2::gfp; rol-6(su1006)]; and TJ356, zIs356 Is[daf-16::daf-16-gfp; for 20 h at 20C in the absence (control) or presence of rol-6]. To obtain synchronous populations worms were either 250 lM juglone or 250 lM juglone plus 250 lM prepared as described [32]. For all experiments synchro- ascorbic acid. Subsequently, worms were washed twice nous populations gained by bleaching were used. Only for with M9 before single nematodes were transferred to 384- lifespan analysis worms were picked and allowed to lay well plates. Sytox Green nucleic acid stain (Invitrogen, eggs to avoid stress conditions. Worms were kept at 20C Karlsruhe, Germany), that penetrates only in cells with compromised plasma membrane and gets fluorescent after in CeHR medium (20; http://www.usacehr.org/cehr_ medium.htm) containing 30% DYT (1.6% casein hydro- DNA binding, was added in a final concentration of 2 lM. Fluorescence was measured at 535 nm over 24 h every lysate, 1% bacto yeast extract, 85.5 mM sodium chloride). OP50 and stocks were provided by the C. elegans Genet- 30 min after excitation at 488 nm using a fluorescence microtiter plate reader (Fluoroskan Ascent FL, Thermo ics Center, CGC, University of Minnesota, Minneapolis, USA. Electron, Dreieich, Germany) that was tempered to 37C. 123 Genes Nutr (2009) 4:59–67 61 Increases of fluorescence values twofold above the back- nitrogen grinding with following sonication. The protein ground indicated the death of the nematodes as verified by content was measured by the BioRad protein assay touch provocation in an independent assay. (BioRad, Munich, Germany). Activities of SOD and cata- lase were determined by UV-absorbance measurements RNA-interference in a Uvicon 930 photometer (Kontron Instruments) as described [1, 22]. In brief, SOD-measurement was based RNA-interference experiments were done as previously on pyrogallol autoxidation at a wavelength of 420 nm, described using bacteria from the Ahringer deletion library which measures overall SOD-activity but does not differ- [16]. Bacterial RNAi feeding strains were seeded on entiate between Cu-, Zn-, and Mn-dependent isoforms. NGM agar plates plus 25 mg/ml carbenicillin and 1 mM Catalase was determined by the rate of disappearance of isopropyl-beta-D-thiogalactopyranoside to induce dsRNA hydrogen peroxide at 240 nm. expression at 37C for 4 h. Before usage plates were cooled down to 20C and L1 worms were put onto plates. Glutathione-levels After reaching L4 or young adult stage worms were washed off the plates with M9 buffer and transferred For GSH-measurements nematodes were incubated for into liquid medium for further experiments. Efficiency 48 h in CeHR-medium with 50 lM FuDR to prevent of RNAi was controlled by RT-PCR. Therefore, mRNA progeny. Subsequent to the incubation worms were washed was extracted from 5,000 to 15,000 synchronized worms several times with M9 buffer and whole worm lysates were (RNAeasy; Qiagen, http://www1.qiagen.com). Unique prepared as described above. GSH and GSSG were quan- primer pairs recognizing only cDNA derived from endog- tified as described [28] by measuring the absorbance at enous daf-16 mRNA were designed to avoid cross-reaction 412 nm after reduction of 5,5 -dithio-2-nitrobenzoic acid with genomic DNA and bacterially generated dsRNA (DTNB, Sigma, Steinheim, Germany). For the GSSG 0 0 and were as follows: 5 GAGACGACTACAAAGGCT3 measurement glutathione has to be derivatized first by 0 0 and 5 GTTCGGGGACGGAAAGA3 . Relative amounts of 2-vinylpyridine following the same measurement like for mRNA-levels for DAF-16 versus the vector control were the total glutathione analysis. The GSH content was cal- 0.15. culated by the difference of total glutathione and the GSSG content. Confocal microscopy Statistical analyses GFP-expression in CL2070 was analyzed at different time points as given in the figure legends and GFP-localization For statistical analysis Gaphpad Prism 4.01 software was in TJ356 after 3 h of incubation in CeHR-medium con- used. Analysis of variance in group comparisons was taining the indicated concentrations of juglone. Thereafter, performed by One-way ANOVA and significance of dif- nematodes were anesthetized by the use of 5 mM levam- ferences between groups were determined by a Tukey’s isole and a drop of the worm pellet was put on a glass slide multiple comparison test. For comparison of survival pre-equipped with a thin layer of 2% agarose gel to avoid curves the Log rank test was used with P values \0.05 desiccation of the worms. Fluorescence was viewed under being significant. Kaplan–Meier survival curves are shown a confocal laser scanning microscope (Leica TCS SP2) in lifespan and heat stress survival figures. with excitations at 488 nm and emissions at 500–530 nm and fluorescence intensities were determined by use of the Leica Confocal Software, version 2.5. Each experiment Results was repeated at least three times and ten worms per group were used in each experiment. Although C. elegans appears as a perfect model to study nutrient-gene-interactions due to the ease of genetic mod- SOD- and catalase-activities ifications and the availability of numerous mutant strains, modifications of its nutritional environment are hard to For activity measurements of SOD and catalase worms achieve when they are grown on agar plates with E. coli as were incubated with or without juglone (control) in CeHR- a food source. Dissolving compounds on agar plates and medium for 1 day or 5 days, respectively, in the presence the bacterial lawn seems not to be the method of choice as of 50 lM 5-fluorodeoxyuridine (FuDR) to prevent pro- the cuticula of the animals limits penetration by simple duction of progeny. Thereafter whole worm lysates were diffusion and ingestion via the bacteria seems also variable. prepared by pelleting the worms before they were resus- When young adult CL2070 nematodes carrying a hsp- pended in native lysis buffer and cracked open by liquid 16.2::gfp transgene were exposed to 100 lM juglone either 123 62 Genes Nutr (2009) 4:59–67 on agar plates with E. coli and the ROS-generator or in despite the fact that median life spans were very similar, liquid axenic medium with dissolved juglone it became maximum lifespan was drastically increased in axenic evident that activation of the hsp-16.2 driven GFP liquid medium (Fig. 2a). This suggests an important role expression is much more consistent and much higher in for bacterial products in limiting the maximal lifespan liquid medium (Fig. 1a). The dose-response curve for the when animals are grown on agar plates with E. coli. activation of the hsp-16.2 promoter was maximal at 4 h Interestingly, CL2070 displayed a highly prolonged subsequent to juglone exposure (Fig. 1b) in a concentration lifespan when compared to wildtype animals in the axenic range of 40–100 lM juglone whereas at higher concentra- culture medium (Fig. 2b) most likely by an integration of tions GFP-expression declined again (Fig. 1c). Moreover, the transgene into lifespan-determining loci of the genome. repeated application of 100 lM juglone every second day When young adult CL2070 were exposed to 100 lM jug- over 8 days always caused GFP-expression to a similar lone their lifespan curves remained unaffected (Fig. 2b), extent (not shown) suggesting that the decline in fluores- indicating that the animals were able to cope with the cence observed after 4 h (Fig. 1b) is due to diminished oxidative stress under these conditions. Concentrations of ROS-levels and not to a down-regulation of the stress 250 lM juglone, however, caused a fast decline in the response during this period of life. numbers of animals alive (Fig. 2b). Previous work in C. elegans suggests that keeping ani- Assuming that the increase in fluorescence in hsp- mals in liquid axenic media slows development, declines 16.2p::gfp transgenic CL2070 animals in the presence of fecundity, increases lifespan, decreases lipid and protein juglone (Fig. 1b, c) was dependent on DAF-16, we deter- stores, and changes gene expression relative to those held mined DAF-16 nuclear translocation in the transgenic on a bacterial diet. Many of these alterations mimic the strain TJ356 that expresses a DAF-16::GFP fusion protein. consequence of dietary restriction and therefore we altered Both, 100 and 250 lM juglone caused GFP-fluorescence to the standard liquid culture medium with respect to an disappear from the cytosol with a concomitant accumula- optimisation of median lifespans for wildtype animals tion of staining in the nuclei (Fig. 3a). Moreover, in the grown in liquid culture as compared to wildtype C. elegans CL2070 strain, juglone-induced hsp-16.2 promoter driven grown on agar plates with E. coli (Fig. 2a). However, GFP expression was completely blocked when DAF-16 liquid medium agar plates B C control *** + juglone *** 150 150 *** ** 100 100 50 50 0 0 0 5 10 15 20 25 incubation time [h] Fig. 1 HSP-16.2 expression as visualized by GFP-fluorescence in a and light microscopy images (a, right panels). Time-dependent GFP- hsp-16.2p::GFP transgenic strain (CL2070). Green fluorescence of fluorescence is shown in (b) when CL2070 nematodes were exposed GFP in CL2070 exposed for 4 h to 100 lM juglone either on agar to 100 lM juglone in liquid axenic medium. Intensities of GFP plates with E. coli as food source (left panel) or in axenic liquid expression in CL2070 cultured in liquid axenic medium for 4 h culture medium (right panel) can be visualized from the fluorescence at various juglone concentrations is shown in (c). *P \ 0.05, images as given in (a), left panels, and the overlays of fluorescence **P \ 0.01, ***P \ 0.001 versus the control control 10 µM 20 µM 40 µM 100µM 150 µM 250µM GFP-fluorescence 2] [arbitrary untis/µm GFP-fluorescence [arbitrary units/µm ] Genes Nutr (2009) 4:59–67 63 1.00 Discussion N2, E. coli N2, CeHR medium 0.75 Caenorhabditis elegans has proven to be a suitable model for studying the genetic control of lifespan. Aging research 0.50 in C. elegans is mainly driven by the availability of the entire genome sequence, the adaptation of RNAi technol- ogy for genome-wide loss-of-function analysis [5] and the 0.25 availability of numerous mutant strains that help to define the role of distinct genes in physiological processes [8]. It 0.00 0 10 20 30 40 50 seems particularly intriguing to take these concepts further time [days] and assess changes in the nutritional environment on basis of different genetic backgrounds. So far, however, studies 1.00 B in C. elegans have almost exclusively utilized feeding CL2070 regimes in which animals are kept on agar plates with an + juglone 100 µM 0.75 E. coli lawn. Metabolism of supplied nutrients or non- + juglone 250 µM nutritional components by the bacteria and production of 0.50 mainly unknown metabolites that might contribute to the biological effects of a dietary ingredient is a concern [33]. 0.25 The use of liquid axenic media on the other hand produce phenotypic changes in development, fecundity, lifespan 0.00 and metabolism when compared to the apparent ‘‘gold 0 25 50 75 100 125 standard’’ of feeding bacteria [10, 33]. It was shown that time [days] these alterations are mainly caused by a dietary restriction and mediated by mechanisms which are distinctly different Fig. 2 Lifespan curves of N2 wildtype animals grown on agar plates from those specified by the insulin/IGF-1 signaling path- with E. coli as a food source in comparison to those measured in liquid axenic medium is shown in (a). Lifespans of CL2070 in axenic way [9, 11]. In the present study we report findings with an liquid medium either in the absence (control) or presence of 100 or optimised liquid axenic medium that is obviously not 250 lM juglone are given in (b). P-values for significances of causing a dietary restriction phenotype as judged on iden- differences of survival curves were P = 0.7907 for 100 lM juglone tical median lifespans of wildtype worms grown on either and P \ 0.0001 for 250 lM juglone versus the untreated CL2070 nematodes agar plates with E. coli or in liquid culture without bacteria. Maximum lifespan, however, was found to be prolonged in the axenic medium. It has been proposed that an increased was knocked-down by RNAi (Fig. 3b). The knock-down of susceptibility to bacterial infections or bacterial products in DAF-16 blocked also the increase in GSH-levels in older animals when fed on bacteria may cause death [7] CL2070 upon exposure of animals to 100 or 250 lMof and the lack of these compromising conditions under juglone (Fig. 4a, b), whereas GSSG-levels remained axenic feeding may cause the extension of maximal life unaffected by juglone (Fig. 4c). Overall, the results indi- span. cate that nuclear DAF-16 is inevitable for a proper ROS We here assessed the effects of the ROS-generator mediated stress response and that the most important juglone supplied per os on C. elegans lifespan in axenic downstream targets that determine resistance to ROS are medium. The use of CL2070 expressing GFP under control the elevated GSH-levels but not the other DAF-16 targets, of the hsp-16.2 promoter [20] allowed the simultaneous such as SOD or catalase that did not change in activity visualization of stress response. CL2070 showed an unex- upon juglone treatment in CL2070 (Fig. 4c, d). pected threefold increase in median, average and maximum In order to demonstrate that the adaptive responses to lifespan compared to N2 wildtype animals in axenic juglone are indeed a consequence of oxidative stress medium. CL2070 transgenic animals were generated by ascorbic acid was applied at 250 lM to scavenge ROS as microinjection of a plasmid carrying the hsp-16.2p::gfp caused by 250 lM juglone. Ascorbic acid blocked the construct along with a rol-6 morphological marker plasmid juglone-induced nuclear translocation of DAF-16, hsp- into the gonads of wildtype C. elegans [20]. Since N2 16.2 promoter activation, and increase in GSH-levels wildtype displayed identical median lifespan in our axenic (Fig. 5a–c). Moreover, ascorbic acid almost completely medium and on agar plates and since the transgene in abolished the survival reducing effect of preincubation CL2070 is chromosomally integrated [20] our findings with 250 lM juglone in a subsequent heat resistance assay suggest an integration of the plasmid into gene sequences (Fig. 5d). that contribute to life span regulation which in turn causes fraction alive fraction alive 64 Genes Nutr (2009) 4:59–67 Fig. 3 HSP-16.2 expression is dependent on DAF-16. In TJ356 evidenced by the lack of GFP-fluorescence in (b). Left panels show DAF-16 localization in the nucleus is increased by 100 and 250 lM the fluorescence images and right panels an overlay of fluorescence juglone (a). Knock-down of DAF-16 by RNA-interference abolished and light microscopic images juglone-induced activation of the hsp-16-2 promoter in CL2070 as is this longevity phenotype of CL2070. Independent on this temperatures [14, 15]. Expression of HSP-16.2 in response basic longevity phenotype of CL2070, the animals show a to the ROS-challenge is mediated by the transcription hormetic stress response allowing the worms to cope factor DAF-16 since knock-down of DAF-16 by RNAi properly with the increased ROS challenge caused by completely abrogated GFP-fluorescence in CL2070. juglone concentrations of up to 100 lM whereas at Moreover, juglone causes DAF-16 translocation from 250 lM premature death was observed. The expression of cytosol into the nucleus and here DAF-16 can promote the small heat shock protein HSP-16.2 increased in a dose transcriptional activation of HSP-16.2 expression. Our dependent fashion as a function of increasing juglone results emphasize the crucial role of DAF-16 in induction concentrations with a maximal response observed at of small HSPs, which in turn can promote longevity and 100 lM juglone. At 250 lM juglone HSP-16.2 driven GFP that this stress response pathway initiated by ROS obvi- fluorescence decreased again in parallel with premature ously follows the same pathways as shown for heat stress death most likely caused by increased oxidative damage [12] or hypertonic stress [18]. One of the major targets of that prevents a further hormetic adaptation. HSP-16.2 HSP-16.2 are enzyme systems that control cellular GSH- levels have been shown to correlate with life expectancy in levels. It has been shown in several mammalian cell lines an isogenic C. elegans population [29] and our data con- that expression of different small HSPs increased the cel- firm that the expression of this heat shock protein can also lular contents of GSH and this response seems essential for act as a good ROS-sensor. the protective activity of these proteins against multiple The HSP-16.2 dose-dependence to juglone exposure stressors [23, 29]. We also observed a two-fold increase in is mimicked by HSP-16.2 adaptations to increasing the levels of GSH that followed the induction of HSP-16.2 123 Genes Nutr (2009) 4:59–67 65 A 30 B 30 C 30 ** + daf-16 RNAi ** 20 20 10 10 10 0 0 0 day 1 day 5 day 1 day 5 day 1 day 5 D E 75 125 CL2070 + 100 µM juglone 50 + 250 µM juglone 0 0 day 1 day 5 day 1 day 5 Fig. 4 GSH-levels but not SOD- or catalase activities are increased (b). GSSG-levels remained unaffected by juglone (c). SOD-activities by juglone in CL2070. Levels of GSH were increased by the ROS- (d) and catalase-activities (e) did not differ significantly between generator (a) with *P \ 0.05, and **P \ 0.01 versus the control, but untreated CL2070 or those treated with 100 or 250 lM juglone DAF-16 RNAi inhibited the juglone-induced increase in GSH-levels CL2070 CL2070 +250 µM juglone/ +250 µM juglone/ +250 µM juglone +250 µM ascorbic acid +250 µM juglone +250 µM ascorbic acid C D 1.00 0.75 CL2070 + 250 µM juglone + 250 µM juglone + 250 µM juglone 0.50 + 250 µM asc. acid ** + 250 µM juglone + 250 µM asc. acid 0.25 0.00 0 5 10 15 day 1 day 5 time (h) Fig. 5 Ascorbic acid prevents juglone-induced effects in CL2070. were all caused by 250 lM juglone. ***P \ 0.001 versus juglone- Ascorbic acid at 250 lM blocks nuclear translocation of DAF-16 in treated nematodes. P-value for significance of difference of survival TJ356 (a), and in CL2070 hsp-16.2p::GFP expression (b), increase in curve of CL2070 treated with 250 lM juglone plus 250 lM ascorbic GSH-levels (c), and reductions in survival under heat-stress (d), that acid and those treated with 250 lM juglone alone was P \ 0.0001 in CL2070 and this response was blocked by DAF-16 are due to de novo synthesis. Known other target genes of RNAi. Since GSSG-levels remained unaltered by juglone- DAF-16 such as sod-3 or ctl-1 however did not show any treatment our results suggest that the increased GSH-levels changes in enzyme activities in response to juglone GSH-level SOD-activity GSH-level [nmol/mg protein] [U/mg protein] [nmol/mg protein] catalase-activity GSH-level [U/mg protein] [nmol/mg protein] fraction alive GSSG-level [nmol/mg protein] 66 Genes Nutr (2009) 4:59–67 Ins/IGF-1 signalling pathway in Caenorhabditis elegans. Exp treatment. Recently it was shown that suppression of sod-3 Gerontol 38:947–954 expression in C. elegans has no or only modest effects on 10. Houthoofd K, Braeckman BP, Lenaerts I, Brys K, De Vreese A, longevity [24, 25]. This is surprising since in long living Van Eygen S, Vanfleteren JR (2002) Axenic growth up-regulates age-1 mutants, in which interrupted insulin signaling cau- mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans. Exp Gerontol 37:1371–1378 ses hyperresistance to oxidative stress both, SOD and 11. Houthoofd K, Braeckman BP, Lenaerts I, Brys K, Matthijssens F, catalase, showed an age-dependent increase that was not De Vreese A, Van Eygen S, Vanfleteren JR (2005) DAF-2 seen in the parental strain [19]. But other studies also failed pathway mutations and food restriction in aging Caenorhabditis to demonstrate changes in gene expression of both anti- elegans differentially affect metabolism. Neurobiol Aging 26: 689–696 oxidative enzymes in response to a short-term exposure to 12. Hsu AL, Murphy CT, Kenyon C (2003) Regulation of aging and 90% oxygen in wildtype, daf-16 or age-1 mutants. This age-related disease by DAF-16 and heat-shock factor. Science suggest as our data also indicate that SOD and catalase do 300:1142–1145 not play a prominent role in the adaptive response to oxi- 13. Ji LL (1999) Antioxidants and oxidative stress in exercise. Proc Soc Exp Biol Med 222:283–292 dative stress [36]. That, however, the responses measured 14. Jones D, Candido EP (1999) Feeding is inhibited by sublethal in C. elegans are indeed dependent on oxidative stress concentrations of toxicants and by heat stress in the nematode was demonstrated by applying ascorbic acid in order to Caenorhabditis elegans: relationship to the cellular stress scavenge the ROS produced by juglone. Ascorbic acid response. J Exp Zool 284:147–157 15. 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Lamitina ST, Strange K (2005) Transcriptional targets of DAF-16 animals to maintain a normal lifespan until the ROS stress insulin signaling pathway protect C. elegans from extreme exceeds a certain threshold of adaptive protective capacity hypertonic stress. Am J Physiol 288:C467–C474 leading to acute toxicity and premature death. 19. Larsen PL (1993) Aging and resistance to oxidative damage in Caenorhabditis elegans. Proc Natl Acad Sci 90:8905–8909 Acknowledgments We thank the Caenorhabditis Genetics Center, 20. Link CD, Cypser JR, Johnson CJ, Johnson TE (1999) Direct University of Minnesota for supplying the Bristol N2, CL2070 and observation of stress response in Caenorhabditis elegans using a TJ356 strains. We acknowledge Mr. A. Stamfort for statistical advice. reporter transgene. Cell Stress Chaperones 4:235–242 This work was supported by the BASF, Ludwigshafen, Germany. 21. 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Feeding a ROS-generator to Caenorhabditis elegans leads to increased expression of small heat shock protein HSP-16.2 and hormesis

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Springer Journals
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Copyright © 2009 by Springer-Verlag
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Biomedicine; Biomedicine general; Gene Function; Nutrition; Human Genetics
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1555-8932
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1865-3499
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10.1007/s12263-009-0113-x
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19252938
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Abstract

Genes Nutr (2009) 4:59–67 DOI 10.1007/s12263-009-0113-x RESEARCH PAPER Feeding a ROS-generator to Caenorhabditis elegans leads to increased expression of small heat shock protein HSP-16.2 and hormesis Kai Hartwig Æ Tanja Heidler Æ Jan Moch Æ Hannelore Daniel Æ Uwe Wenzel Received: 16 October 2008 / Accepted: 10 February 2009 / Published online: 28 February 2009 Springer-Verlag 2009 Abstract Reactive oxygen species (ROS) are thought to Introduction be a driving force in the aging process. In transgenic Cae- norhabditis elegans expressing green fluorescent protein The exposure to high amounts of reactive oxygen species (GFP) under control of the hsp-16.2 promoter (CL2070) 100 (ROS), as occurs in many tissues, e.g. by heavy physical lM of the ROS-generator juglone induced GFP-expression. exercise, is answered by prominent adaptive responses of This was associated with translocation of DAF-16 to the the antioxidant defense system, which is of vital impor- nucleus as visualized in a transgenic strain expressing a tance in the protection against oxidative stress [13]. Small DAF-16::GFP fusion protein (TJ356) and with increased heat-shock proteins (HSPs), a family of low-molecular cellular levels of reduced glutathione. RNA-interference for weight polypeptides that are highly conserved from DAF-16 in CL2070 blocked the juglone-induced HSP-16.2 yeast to humans [6, 34], appear of general importance expression and the increase in glutathione levels. Higher for adaptations as their expression correlates usually concentrations of juglone did not further increase the well with the presence of various stressors [21, 31]. In adaptive responses but caused premature death, indicating the nematode Caenorhabditis elegans levels of small hormetic adaptations unless the stressor exceeds the intrin- HSP-16.2 on the first day of adult life predicts as much as sic protective capacity. The addition of the ROS-scavenger a fourfold variation in subsequent survival [29]. This ascorbic acid finally blocked lifespan reductions and all of correlation was highly specific for HSP-16.2 since the the adaptations to juglone stressing that ROS are indeed the induction of other genes normally activated by stress did molecular species that require protective response. not show immediate relationship to longevity [29]. Moreover, HSP-16.2 expression was identified as a good Keywords Caenorhabditis elegans  Hormesis  predictor of the ability to withstand a lethal thermal stress Reactive oxygen species  Aging  Heat shock protein  [29] and its levels correlate well with the hormetic effects Glutathione in response to heat treatment [26]. Mechanistically, HSP- 16 could affect lifespan by reducing oxidative stress through raising the pool of reduced glutathione (GSH) in the worm as suggested by Walker and Lithgow [35]. In a similar fashion HSP-27 is thought to mediate its effects in K. Hartwig  T. Heidler  H. Daniel Department of Food and Nutrition, murine cells [23, 27]. This protection against oxidative Molecular Nutrition Unit, stress appears to be accomplished by small HSPs through Technical University of Munich, a glucose-6-phosphate dehydrogenase-dependent increase Am Forum 5, 85350 Freising, Germany in NADPH generation needed to maintain GSH in its J. Moch  U. Wenzel (&) reduced form via the GSSG-reductase and by using this Molecular Nutrition Research, redox modulator as an essential cofactor of their in vivo Interdisciplinary Research Center, chaperone activity against oxidized proteins [27]. This Justus-Liebig-University of Giessen, function of HSP-16.2 in stress resistance and longevity is Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany embedded into the insulin/IGF-1 signaling pathway that e-mail: [email protected] 123 60 Genes Nutr (2009) 4:59–67 has been shown in many studies to be a central deter- Lifespan analysis minant of lifespan in C. elegans [30]. The DAF-16 transcription factor, a downstream target of the insulin/ Synchronous populations were obtained by allowing 50 IGF-1 receptor DAF-2 that translocates into the nucleus hermaphrodites to lay eggs for 3 h. Eggs were placed on 50 mm NGM plates seeded with E. coli OP50 (40–50 per in the absence of insulin/IGF-1 signaling, is essential for maximal HSP-16 expression and for lifespan extension plate, 80–300 individuals per experiment) and grown at 20C until L4 larval stage. Each population was examined conferred by it [35]. Besides the hsp-16.2 gene, however, sod-3 coding for a superoxide dismutase (SOD) and ctl-1 for synchrony at the beginning of the reproductive period. Lifespan analysis on agar plates with E. coli as the food and ctl-2 coding for catalase isoforms are further tran- scriptional targets of DAF-16 that contribute to the source was carried out according to standard protocols longevity phenotype of daf-2 mutants, probably by pre- [17]. After reaching the young adult stage worms were venting or repairing oxidative macromolecular damage, transferred to new NGM agar plates with growth arrested and knock-down of those genes by RNA-interference E. coli, which were treated before with 100 lg/ml ampi- (RNAi) reduces the lifespan in daf-2 mutants by 10–20% cillin. Worms were monitored every day and transferred to [24, 25]. fresh plates every third day. They were scored as dead when no response to a mechanical stimulus or no pharynx In the present study we assessed whether application of juglone, a generator of superoxide anion radicals [4], contraction was visible. Animals that crawled off the plates were replaced by a new worm from a substitution plate. delivered via the oral route triggers the stress response in C. elegans and in how far the adaptations to juglone enable For lifespan analysis in liquid axenic medium L4 larvae the maintenance of a normal lifespan. Nematodes were were transferred into wells containing CeHR medium and grown from the L4 stage on in an adapted liquid axenic were distributed into new wells after reaching young adult medium to assure the proper oral uptake and transport of stage. Juglone was applied in 92% ethanol, 8% Tween 80 juglone through pumping in the worm pharynx posteriorly (v/v) and diluted 1:100 in the final medium. Controls were [2]. Induction of HSP-16.2 was estimated by the expression always exposed to the same amount of solvent. Worms of green fluorescent protein (GFP) in a hsp-16.2p::gfp were moved to wells containing fresh medium and com- transgenic strain (CL2070) and translocation of DAF-16 pounds every third day with a glass capillary and scored as dead when they did not respond to a mechanical stimulus was visualized using a daf-16::gfp transgenic strain (TJ356). Activities of SOD, catalase and levels of reduced or no more pharynx contraction was seen. Experiments were started with 70 worms per group and ten worms as a (GSH) and oxidized (GSSG) glutathione were determined by UV-absorbance based assays. reserve for lost or bagged individuals. Stress resistance Methods For measuring resistance of CL2070 to heat stress nema- Nematode strains, maintenance and mass culture todes were grown in NGM medium at 20C. Therefore, approximately ten L1 larvae stage worms in 10 llM9 Nematodes were grown on NGM agar plates seeded buffer were dispensed to each well of a 96-well flat- bottomed tissue culture plate together with 40 llof with E. coli strain OP50 as described [3] and were kept at 20C. Strains used in this study were: N2, variation resuspended OP50 bacterial culture with OD = 1. After reaching the young adult stage nematodes were incubated bristol (wildtype); CL2070, dvIs70 Is[hsp-16.2::gfp; rol-6(su1006)]; and TJ356, zIs356 Is[daf-16::daf-16-gfp; for 20 h at 20C in the absence (control) or presence of rol-6]. To obtain synchronous populations worms were either 250 lM juglone or 250 lM juglone plus 250 lM prepared as described [32]. For all experiments synchro- ascorbic acid. Subsequently, worms were washed twice nous populations gained by bleaching were used. Only for with M9 before single nematodes were transferred to 384- lifespan analysis worms were picked and allowed to lay well plates. Sytox Green nucleic acid stain (Invitrogen, eggs to avoid stress conditions. Worms were kept at 20C Karlsruhe, Germany), that penetrates only in cells with compromised plasma membrane and gets fluorescent after in CeHR medium (20; http://www.usacehr.org/cehr_ medium.htm) containing 30% DYT (1.6% casein hydro- DNA binding, was added in a final concentration of 2 lM. Fluorescence was measured at 535 nm over 24 h every lysate, 1% bacto yeast extract, 85.5 mM sodium chloride). OP50 and stocks were provided by the C. elegans Genet- 30 min after excitation at 488 nm using a fluorescence microtiter plate reader (Fluoroskan Ascent FL, Thermo ics Center, CGC, University of Minnesota, Minneapolis, USA. Electron, Dreieich, Germany) that was tempered to 37C. 123 Genes Nutr (2009) 4:59–67 61 Increases of fluorescence values twofold above the back- nitrogen grinding with following sonication. The protein ground indicated the death of the nematodes as verified by content was measured by the BioRad protein assay touch provocation in an independent assay. (BioRad, Munich, Germany). Activities of SOD and cata- lase were determined by UV-absorbance measurements RNA-interference in a Uvicon 930 photometer (Kontron Instruments) as described [1, 22]. In brief, SOD-measurement was based RNA-interference experiments were done as previously on pyrogallol autoxidation at a wavelength of 420 nm, described using bacteria from the Ahringer deletion library which measures overall SOD-activity but does not differ- [16]. Bacterial RNAi feeding strains were seeded on entiate between Cu-, Zn-, and Mn-dependent isoforms. NGM agar plates plus 25 mg/ml carbenicillin and 1 mM Catalase was determined by the rate of disappearance of isopropyl-beta-D-thiogalactopyranoside to induce dsRNA hydrogen peroxide at 240 nm. expression at 37C for 4 h. Before usage plates were cooled down to 20C and L1 worms were put onto plates. Glutathione-levels After reaching L4 or young adult stage worms were washed off the plates with M9 buffer and transferred For GSH-measurements nematodes were incubated for into liquid medium for further experiments. Efficiency 48 h in CeHR-medium with 50 lM FuDR to prevent of RNAi was controlled by RT-PCR. Therefore, mRNA progeny. Subsequent to the incubation worms were washed was extracted from 5,000 to 15,000 synchronized worms several times with M9 buffer and whole worm lysates were (RNAeasy; Qiagen, http://www1.qiagen.com). Unique prepared as described above. GSH and GSSG were quan- primer pairs recognizing only cDNA derived from endog- tified as described [28] by measuring the absorbance at enous daf-16 mRNA were designed to avoid cross-reaction 412 nm after reduction of 5,5 -dithio-2-nitrobenzoic acid with genomic DNA and bacterially generated dsRNA (DTNB, Sigma, Steinheim, Germany). For the GSSG 0 0 and were as follows: 5 GAGACGACTACAAAGGCT3 measurement glutathione has to be derivatized first by 0 0 and 5 GTTCGGGGACGGAAAGA3 . Relative amounts of 2-vinylpyridine following the same measurement like for mRNA-levels for DAF-16 versus the vector control were the total glutathione analysis. The GSH content was cal- 0.15. culated by the difference of total glutathione and the GSSG content. Confocal microscopy Statistical analyses GFP-expression in CL2070 was analyzed at different time points as given in the figure legends and GFP-localization For statistical analysis Gaphpad Prism 4.01 software was in TJ356 after 3 h of incubation in CeHR-medium con- used. Analysis of variance in group comparisons was taining the indicated concentrations of juglone. Thereafter, performed by One-way ANOVA and significance of dif- nematodes were anesthetized by the use of 5 mM levam- ferences between groups were determined by a Tukey’s isole and a drop of the worm pellet was put on a glass slide multiple comparison test. For comparison of survival pre-equipped with a thin layer of 2% agarose gel to avoid curves the Log rank test was used with P values \0.05 desiccation of the worms. Fluorescence was viewed under being significant. Kaplan–Meier survival curves are shown a confocal laser scanning microscope (Leica TCS SP2) in lifespan and heat stress survival figures. with excitations at 488 nm and emissions at 500–530 nm and fluorescence intensities were determined by use of the Leica Confocal Software, version 2.5. Each experiment Results was repeated at least three times and ten worms per group were used in each experiment. Although C. elegans appears as a perfect model to study nutrient-gene-interactions due to the ease of genetic mod- SOD- and catalase-activities ifications and the availability of numerous mutant strains, modifications of its nutritional environment are hard to For activity measurements of SOD and catalase worms achieve when they are grown on agar plates with E. coli as were incubated with or without juglone (control) in CeHR- a food source. Dissolving compounds on agar plates and medium for 1 day or 5 days, respectively, in the presence the bacterial lawn seems not to be the method of choice as of 50 lM 5-fluorodeoxyuridine (FuDR) to prevent pro- the cuticula of the animals limits penetration by simple duction of progeny. Thereafter whole worm lysates were diffusion and ingestion via the bacteria seems also variable. prepared by pelleting the worms before they were resus- When young adult CL2070 nematodes carrying a hsp- pended in native lysis buffer and cracked open by liquid 16.2::gfp transgene were exposed to 100 lM juglone either 123 62 Genes Nutr (2009) 4:59–67 on agar plates with E. coli and the ROS-generator or in despite the fact that median life spans were very similar, liquid axenic medium with dissolved juglone it became maximum lifespan was drastically increased in axenic evident that activation of the hsp-16.2 driven GFP liquid medium (Fig. 2a). This suggests an important role expression is much more consistent and much higher in for bacterial products in limiting the maximal lifespan liquid medium (Fig. 1a). The dose-response curve for the when animals are grown on agar plates with E. coli. activation of the hsp-16.2 promoter was maximal at 4 h Interestingly, CL2070 displayed a highly prolonged subsequent to juglone exposure (Fig. 1b) in a concentration lifespan when compared to wildtype animals in the axenic range of 40–100 lM juglone whereas at higher concentra- culture medium (Fig. 2b) most likely by an integration of tions GFP-expression declined again (Fig. 1c). Moreover, the transgene into lifespan-determining loci of the genome. repeated application of 100 lM juglone every second day When young adult CL2070 were exposed to 100 lM jug- over 8 days always caused GFP-expression to a similar lone their lifespan curves remained unaffected (Fig. 2b), extent (not shown) suggesting that the decline in fluores- indicating that the animals were able to cope with the cence observed after 4 h (Fig. 1b) is due to diminished oxidative stress under these conditions. Concentrations of ROS-levels and not to a down-regulation of the stress 250 lM juglone, however, caused a fast decline in the response during this period of life. numbers of animals alive (Fig. 2b). Previous work in C. elegans suggests that keeping ani- Assuming that the increase in fluorescence in hsp- mals in liquid axenic media slows development, declines 16.2p::gfp transgenic CL2070 animals in the presence of fecundity, increases lifespan, decreases lipid and protein juglone (Fig. 1b, c) was dependent on DAF-16, we deter- stores, and changes gene expression relative to those held mined DAF-16 nuclear translocation in the transgenic on a bacterial diet. Many of these alterations mimic the strain TJ356 that expresses a DAF-16::GFP fusion protein. consequence of dietary restriction and therefore we altered Both, 100 and 250 lM juglone caused GFP-fluorescence to the standard liquid culture medium with respect to an disappear from the cytosol with a concomitant accumula- optimisation of median lifespans for wildtype animals tion of staining in the nuclei (Fig. 3a). Moreover, in the grown in liquid culture as compared to wildtype C. elegans CL2070 strain, juglone-induced hsp-16.2 promoter driven grown on agar plates with E. coli (Fig. 2a). However, GFP expression was completely blocked when DAF-16 liquid medium agar plates B C control *** + juglone *** 150 150 *** ** 100 100 50 50 0 0 0 5 10 15 20 25 incubation time [h] Fig. 1 HSP-16.2 expression as visualized by GFP-fluorescence in a and light microscopy images (a, right panels). Time-dependent GFP- hsp-16.2p::GFP transgenic strain (CL2070). Green fluorescence of fluorescence is shown in (b) when CL2070 nematodes were exposed GFP in CL2070 exposed for 4 h to 100 lM juglone either on agar to 100 lM juglone in liquid axenic medium. Intensities of GFP plates with E. coli as food source (left panel) or in axenic liquid expression in CL2070 cultured in liquid axenic medium for 4 h culture medium (right panel) can be visualized from the fluorescence at various juglone concentrations is shown in (c). *P \ 0.05, images as given in (a), left panels, and the overlays of fluorescence **P \ 0.01, ***P \ 0.001 versus the control control 10 µM 20 µM 40 µM 100µM 150 µM 250µM GFP-fluorescence 2] [arbitrary untis/µm GFP-fluorescence [arbitrary units/µm ] Genes Nutr (2009) 4:59–67 63 1.00 Discussion N2, E. coli N2, CeHR medium 0.75 Caenorhabditis elegans has proven to be a suitable model for studying the genetic control of lifespan. Aging research 0.50 in C. elegans is mainly driven by the availability of the entire genome sequence, the adaptation of RNAi technol- ogy for genome-wide loss-of-function analysis [5] and the 0.25 availability of numerous mutant strains that help to define the role of distinct genes in physiological processes [8]. It 0.00 0 10 20 30 40 50 seems particularly intriguing to take these concepts further time [days] and assess changes in the nutritional environment on basis of different genetic backgrounds. So far, however, studies 1.00 B in C. elegans have almost exclusively utilized feeding CL2070 regimes in which animals are kept on agar plates with an + juglone 100 µM 0.75 E. coli lawn. Metabolism of supplied nutrients or non- + juglone 250 µM nutritional components by the bacteria and production of 0.50 mainly unknown metabolites that might contribute to the biological effects of a dietary ingredient is a concern [33]. 0.25 The use of liquid axenic media on the other hand produce phenotypic changes in development, fecundity, lifespan 0.00 and metabolism when compared to the apparent ‘‘gold 0 25 50 75 100 125 standard’’ of feeding bacteria [10, 33]. It was shown that time [days] these alterations are mainly caused by a dietary restriction and mediated by mechanisms which are distinctly different Fig. 2 Lifespan curves of N2 wildtype animals grown on agar plates from those specified by the insulin/IGF-1 signaling path- with E. coli as a food source in comparison to those measured in liquid axenic medium is shown in (a). Lifespans of CL2070 in axenic way [9, 11]. In the present study we report findings with an liquid medium either in the absence (control) or presence of 100 or optimised liquid axenic medium that is obviously not 250 lM juglone are given in (b). P-values for significances of causing a dietary restriction phenotype as judged on iden- differences of survival curves were P = 0.7907 for 100 lM juglone tical median lifespans of wildtype worms grown on either and P \ 0.0001 for 250 lM juglone versus the untreated CL2070 nematodes agar plates with E. coli or in liquid culture without bacteria. Maximum lifespan, however, was found to be prolonged in the axenic medium. It has been proposed that an increased was knocked-down by RNAi (Fig. 3b). The knock-down of susceptibility to bacterial infections or bacterial products in DAF-16 blocked also the increase in GSH-levels in older animals when fed on bacteria may cause death [7] CL2070 upon exposure of animals to 100 or 250 lMof and the lack of these compromising conditions under juglone (Fig. 4a, b), whereas GSSG-levels remained axenic feeding may cause the extension of maximal life unaffected by juglone (Fig. 4c). Overall, the results indi- span. cate that nuclear DAF-16 is inevitable for a proper ROS We here assessed the effects of the ROS-generator mediated stress response and that the most important juglone supplied per os on C. elegans lifespan in axenic downstream targets that determine resistance to ROS are medium. The use of CL2070 expressing GFP under control the elevated GSH-levels but not the other DAF-16 targets, of the hsp-16.2 promoter [20] allowed the simultaneous such as SOD or catalase that did not change in activity visualization of stress response. CL2070 showed an unex- upon juglone treatment in CL2070 (Fig. 4c, d). pected threefold increase in median, average and maximum In order to demonstrate that the adaptive responses to lifespan compared to N2 wildtype animals in axenic juglone are indeed a consequence of oxidative stress medium. CL2070 transgenic animals were generated by ascorbic acid was applied at 250 lM to scavenge ROS as microinjection of a plasmid carrying the hsp-16.2p::gfp caused by 250 lM juglone. Ascorbic acid blocked the construct along with a rol-6 morphological marker plasmid juglone-induced nuclear translocation of DAF-16, hsp- into the gonads of wildtype C. elegans [20]. Since N2 16.2 promoter activation, and increase in GSH-levels wildtype displayed identical median lifespan in our axenic (Fig. 5a–c). Moreover, ascorbic acid almost completely medium and on agar plates and since the transgene in abolished the survival reducing effect of preincubation CL2070 is chromosomally integrated [20] our findings with 250 lM juglone in a subsequent heat resistance assay suggest an integration of the plasmid into gene sequences (Fig. 5d). that contribute to life span regulation which in turn causes fraction alive fraction alive 64 Genes Nutr (2009) 4:59–67 Fig. 3 HSP-16.2 expression is dependent on DAF-16. In TJ356 evidenced by the lack of GFP-fluorescence in (b). Left panels show DAF-16 localization in the nucleus is increased by 100 and 250 lM the fluorescence images and right panels an overlay of fluorescence juglone (a). Knock-down of DAF-16 by RNA-interference abolished and light microscopic images juglone-induced activation of the hsp-16-2 promoter in CL2070 as is this longevity phenotype of CL2070. Independent on this temperatures [14, 15]. Expression of HSP-16.2 in response basic longevity phenotype of CL2070, the animals show a to the ROS-challenge is mediated by the transcription hormetic stress response allowing the worms to cope factor DAF-16 since knock-down of DAF-16 by RNAi properly with the increased ROS challenge caused by completely abrogated GFP-fluorescence in CL2070. juglone concentrations of up to 100 lM whereas at Moreover, juglone causes DAF-16 translocation from 250 lM premature death was observed. The expression of cytosol into the nucleus and here DAF-16 can promote the small heat shock protein HSP-16.2 increased in a dose transcriptional activation of HSP-16.2 expression. Our dependent fashion as a function of increasing juglone results emphasize the crucial role of DAF-16 in induction concentrations with a maximal response observed at of small HSPs, which in turn can promote longevity and 100 lM juglone. At 250 lM juglone HSP-16.2 driven GFP that this stress response pathway initiated by ROS obvi- fluorescence decreased again in parallel with premature ously follows the same pathways as shown for heat stress death most likely caused by increased oxidative damage [12] or hypertonic stress [18]. One of the major targets of that prevents a further hormetic adaptation. HSP-16.2 HSP-16.2 are enzyme systems that control cellular GSH- levels have been shown to correlate with life expectancy in levels. It has been shown in several mammalian cell lines an isogenic C. elegans population [29] and our data con- that expression of different small HSPs increased the cel- firm that the expression of this heat shock protein can also lular contents of GSH and this response seems essential for act as a good ROS-sensor. the protective activity of these proteins against multiple The HSP-16.2 dose-dependence to juglone exposure stressors [23, 29]. We also observed a two-fold increase in is mimicked by HSP-16.2 adaptations to increasing the levels of GSH that followed the induction of HSP-16.2 123 Genes Nutr (2009) 4:59–67 65 A 30 B 30 C 30 ** + daf-16 RNAi ** 20 20 10 10 10 0 0 0 day 1 day 5 day 1 day 5 day 1 day 5 D E 75 125 CL2070 + 100 µM juglone 50 + 250 µM juglone 0 0 day 1 day 5 day 1 day 5 Fig. 4 GSH-levels but not SOD- or catalase activities are increased (b). GSSG-levels remained unaffected by juglone (c). SOD-activities by juglone in CL2070. Levels of GSH were increased by the ROS- (d) and catalase-activities (e) did not differ significantly between generator (a) with *P \ 0.05, and **P \ 0.01 versus the control, but untreated CL2070 or those treated with 100 or 250 lM juglone DAF-16 RNAi inhibited the juglone-induced increase in GSH-levels CL2070 CL2070 +250 µM juglone/ +250 µM juglone/ +250 µM juglone +250 µM ascorbic acid +250 µM juglone +250 µM ascorbic acid C D 1.00 0.75 CL2070 + 250 µM juglone + 250 µM juglone + 250 µM juglone 0.50 + 250 µM asc. acid ** + 250 µM juglone + 250 µM asc. acid 0.25 0.00 0 5 10 15 day 1 day 5 time (h) Fig. 5 Ascorbic acid prevents juglone-induced effects in CL2070. were all caused by 250 lM juglone. ***P \ 0.001 versus juglone- Ascorbic acid at 250 lM blocks nuclear translocation of DAF-16 in treated nematodes. P-value for significance of difference of survival TJ356 (a), and in CL2070 hsp-16.2p::GFP expression (b), increase in curve of CL2070 treated with 250 lM juglone plus 250 lM ascorbic GSH-levels (c), and reductions in survival under heat-stress (d), that acid and those treated with 250 lM juglone alone was P \ 0.0001 in CL2070 and this response was blocked by DAF-16 are due to de novo synthesis. Known other target genes of RNAi. Since GSSG-levels remained unaltered by juglone- DAF-16 such as sod-3 or ctl-1 however did not show any treatment our results suggest that the increased GSH-levels changes in enzyme activities in response to juglone GSH-level SOD-activity GSH-level [nmol/mg protein] [U/mg protein] [nmol/mg protein] catalase-activity GSH-level [U/mg protein] [nmol/mg protein] fraction alive GSSG-level [nmol/mg protein] 66 Genes Nutr (2009) 4:59–67 Ins/IGF-1 signalling pathway in Caenorhabditis elegans. Exp treatment. Recently it was shown that suppression of sod-3 Gerontol 38:947–954 expression in C. elegans has no or only modest effects on 10. Houthoofd K, Braeckman BP, Lenaerts I, Brys K, De Vreese A, longevity [24, 25]. This is surprising since in long living Van Eygen S, Vanfleteren JR (2002) Axenic growth up-regulates age-1 mutants, in which interrupted insulin signaling cau- mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans. Exp Gerontol 37:1371–1378 ses hyperresistance to oxidative stress both, SOD and 11. Houthoofd K, Braeckman BP, Lenaerts I, Brys K, Matthijssens F, catalase, showed an age-dependent increase that was not De Vreese A, Van Eygen S, Vanfleteren JR (2005) DAF-2 seen in the parental strain [19]. But other studies also failed pathway mutations and food restriction in aging Caenorhabditis to demonstrate changes in gene expression of both anti- elegans differentially affect metabolism. Neurobiol Aging 26: 689–696 oxidative enzymes in response to a short-term exposure to 12. Hsu AL, Murphy CT, Kenyon C (2003) Regulation of aging and 90% oxygen in wildtype, daf-16 or age-1 mutants. This age-related disease by DAF-16 and heat-shock factor. Science suggest as our data also indicate that SOD and catalase do 300:1142–1145 not play a prominent role in the adaptive response to oxi- 13. Ji LL (1999) Antioxidants and oxidative stress in exercise. Proc Soc Exp Biol Med 222:283–292 dative stress [36]. That, however, the responses measured 14. Jones D, Candido EP (1999) Feeding is inhibited by sublethal in C. elegans are indeed dependent on oxidative stress concentrations of toxicants and by heat stress in the nematode was demonstrated by applying ascorbic acid in order to Caenorhabditis elegans: relationship to the cellular stress scavenge the ROS produced by juglone. Ascorbic acid response. J Exp Zool 284:147–157 15. 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Lamitina ST, Strange K (2005) Transcriptional targets of DAF-16 animals to maintain a normal lifespan until the ROS stress insulin signaling pathway protect C. elegans from extreme exceeds a certain threshold of adaptive protective capacity hypertonic stress. Am J Physiol 288:C467–C474 leading to acute toxicity and premature death. 19. Larsen PL (1993) Aging and resistance to oxidative damage in Caenorhabditis elegans. Proc Natl Acad Sci 90:8905–8909 Acknowledgments We thank the Caenorhabditis Genetics Center, 20. Link CD, Cypser JR, Johnson CJ, Johnson TE (1999) Direct University of Minnesota for supplying the Bristol N2, CL2070 and observation of stress response in Caenorhabditis elegans using a TJ356 strains. We acknowledge Mr. A. Stamfort for statistical advice. reporter transgene. Cell Stress Chaperones 4:235–242 This work was supported by the BASF, Ludwigshafen, Germany. 21. 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Published: Feb 28, 2009

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