Manganous ion supplementation accelerates wild type development, enhances stress resistance, and rescues the life span of a short–lived Caenorhabditis elegans mutant

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Manganous ion supplementation accelerates wild type development, enhances stress resistance, and rescues the life span of a short–lived Caenorhabditis elegans mutant
  Original Contribution Manganous ion supplementation accelerates wild type development,enhances stress resistance, and rescues the life span of a short  – lived Caenorhabditis elegans  mutant  Yi-Ting Lin, Hanh Hoang, Scott I. Hsieh, Natalie Rangel, Amanda L. Foster  1 , James N. Sampayo 1 ,Gordon J. Lithgow 1 , Chandra Srinivasan ⁎  Department of Chemistry and Biochemistry, California State University, Fullerton, Fullerton, CA 92834, USA Received 19 April 2005; revised 21 October 2005; accepted 8 November 2005Available online 12 December 2005 Abstract Relative to iron and copper we know very little about the cellular roles of manganese. Some studies claim that manganese acts as a radicalscavenger in unicellular organisms, while there have been other reports that manganese causes Parkinson ’ s disease-like syndrome, DNAfragmentation, and interferes with cellular energy production. The goal of this study was to uncover if manganese has any free radical scavenging properties in the complex multicellular organism,  Caenorhabditis elegans . We measured internal manganese in supplemented worms usinginductively coupled plasma mass spectrometry (ICP-MS) and the data obtained suggest that manganese supplemented to the growth medium istaken up by the worms. We found that manganese did not appear to be toxic as supplementation did not negatively effect development or fertility.In fact, supplementation at higher levels accelerated development and increased total fertility of wild type worms by 16%. Manganese-supplemented wild type worms were found to be thermotolerant and, under certain conditions, long-lived. In addition, the oxidatively challenged C. elegans  strain  mev-1 ’ s short life span was significantly increased after manganese supplementation. Although manganese appears to be beneficial to  C. elegans , the mode of action remains unclear. Manganese may work directly as a free radical scavenger, as it has been postulated todo so in unicellular organisms, or may work indirectly by up regulating several protective factors.© 2005 Elsevier Inc. All rights reserved.  Keywords:  Aging; Oxidative stress; Antioxidant; Superoxide dismutase; Free radicals; Manganese;  Caenorhabditis elegans ; Thermotolerance; Heat shock  Introduction Manganese (Mn) is an essential ultratrace element similar tochromium,molybdenum,andcobalt.Itisneededforawidevarietyof physiological processes ranging from the regulation of repro-duction to normal brain function. It is also required for severalenzymes including arginase, pyruvate carboxylase, glycosyl trans-ferase,  E. coli  aminopeptidase, and the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD). Mn canexist in various oxidation states ranging from  − 3 to +7, with +2oxidation state being the most predominant in biological systems.Unlike the well-studied transition metal ions, copper, iron, andzinc, not much is known about ionic manganese in vivo, althoughseveral recent studies have focused on trying to understand the biological chemistry of manganese [1].Ionic manganese appears to have free radical scavenging properties as demonstrated by the fact that aerobic growthdefects of several SOD-null bacterial species can be rescued by Mn(II) supplementation [2]. In the aerobic organism  Lacto-bacillus plantarum , which lacks SOD, ionic manganese levelsare high (30 mM), and it is thought that manganese complexedto intracellular high molecular weight polyphosphate might  provide a defense against superoxide [3]. In bacterial systems,low molecular weight manganese complexes are thought to beworking in conjunction with antioxidant enzymes such as SODand catalase to provide a defense against oxygen radicals [2].Comparison of several lactic acid bacterial species indicates that  Free Radical Biology & Medicine 40 (2006) 1185 –  Abbreviations:  ROS, reactive oxygen species; SOD, superoxide dismutase;MnSOD, manganese-containing SOD; CuZnSOD, copper, zinc-containingSOD; ICP-MS, inductively coupled plasma mass spectrometry; Mn, ionicmanganese; FUDR, 5-fluoro-2-deoxyuridine; WT (or N2), wild type worm. ⁎  Corresponding author. Fax: +1 714 278 5316.  E-mail address: (C. Srinivasan). 1 The Buck Institute, 8001 Redwood Blvd., Novato, CA 94945, USA.0891-5849/$ - see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2005.11.007  when an organism lacks detectable SOD activity they requirehigher levels of manganese than organisms that have SODactivity [4]. A recent study showed that   Deinococcus radio-durans  that are highly resistant to ionizing gamma radiationhave high manganese, linking intracellular manganese accumu-lation that is independent of MnSOD activity, in defense against radiation-mediated production of reactive oxygen species(ROS) [5]. In baker  ’ s yeast, growth of CuZnSOD knockoutsin media containing millimolar levels of manganese rescues all phenotypes of the compromised knockout strain [6] even in theabsence of MnSOD, suggesting that the manganese rescue isalso independent of MnSOD activity in yeast. While redox-active metals such as Fe(II) can accelerate lipid peroxidation,ionic manganese (10 – 100  μ M) has been shown to inhibit lipid peroxidation in rat liver microsomes [7]. Also, several knownmanganese complexes including the manganese salen and man-ganese bis(cyclohexylpyridine)-substituted macro cyclic ligandhave shown promise as possible SOD mimics [8 – 10]. Studieshave shown that these complexes are as effective as SODenzymes in detoxifying superoxide under some experimentalconditions [11]. Some of these complexes also have marginalcatalase activity in addition to their SOD-like activity [9].Despite several reports suggesting the beneficial effects of manganese inunicellularorganisms,itiswellknownthatchronicexposure to high atmospheric levels of manganese is toxic. Re-search shows that an overload of manganese has been documen-ted to cause the disease  “ manganism, ”  which has Parkinson's-like symptoms [12,13]. Manganese is also known to cause cell death,but itisunclear astowhether thisisprovokedbyapoptosisor necrosis. Although Mn-treated cells have activated classicalapoptosis signaling pathways, Mn also interferes with mitochon-drial function as it inactivates complex I of the electron transport chain, 4Fe – 4S cluster containing aconitase [14], and F1-ATPase,all of which are involved in cellular energy production. It has been proposed that this Mn-induced cell death could eventually be due to necrosis even if apoptosis signaling is initiated [12].It is evident that redox-active transition metals like copper and iron in high concentrations can cause damage to various biomolecules through Fenton chemistry. Although manganesehas not been shown to participate in Fenton-type reactions that lead to the generation of hydroxyl radicals, it is neverthelessintriguing to see that in some unicellular organisms, ionic man-ganese can compensate for lack of cellular antioxidant defensesor provide protection against ROS-producing agents. Since allof the studies exploring the radical scavenging function of manganese in vivo thus far have been carried out in unicellular organisms, we sought to examine the role of ionic manganese ina multicellular organism.  C. elegans  provide a valuable systemto address the pro or the antioxidant role for ionic manganese asa number of genetic mutants with altered levels of oxidativestress resistance are available.  mev-1  encodes a subunit of suc-cinate-coenzyme Q oxidoreductase in complex II of the electrontransport chain and mutation results in elevated levels of super-oxide [15,16]. This strain is significantly short-lived compared to wild type, with several phenotypes consistent with elevatedoxidative stress such as sensitivity to the superoxide generator  paraquat or high oxygen [17]. The availability of this well-characterizedstrainprovidesausefultoolfortestingcompoundsfor antioxidant properties [18 – 20].To uncover if manganese has a pro or antioxidant property invivo we provided varying levels of manganese to the growthmedium to see how manganese supplementation affects  C.elegans  development, fertility, thermal stress resistance, andlife span. Our results indicate that ionic manganese is clearlynot toxic to the worms (up to 1 mM) as it does not decrease thelife span or the fertility of the worms. In our study we also sawan increase in mean life span of a manganese supplementedshort-lived  mev-1  strain, and under some experimental condi-tions in the wild type strain also. In addition, we saw anenhanced resistance to thermal stress in wild type  C. elegans ,suggesting a beneficial role for Mn in this higher organism. Materials and methods  Reagents, media, and worm growthC. elegans  strains used in this study were wild type (N2)and  mev-1  (TK22  Kn1 ) and were obtained from the  C. ele- gans  Genetic Stock Center (MN). Worm strains were main-tained on nematode growth media (NGM) plates with an  E.coli  OP50 lawn at 22°C and standard worm plate cultureconditions and techniques were used. The OP50 strain usedin this study was obtained from Dr. Catherine Clarke ’ s Lab-oratory (UCLA). Manganese sulfate (MnSO 4 ) was the sourceof Mn(II) ions in our supplementation studies and it was purchased from Aldrich (99.99+% pure). A 1 M stock solu-tion was prepared by dissolving the salt in deionized water,sterilized by filtering, and this solution was stored at roomtemperature until needed. Calculated amounts of this stock solution were added to the molten standard NGM agar prior to pouring to obtain NGM plates containing Mn(II). 5-Fluoro-2-deoxyuridine (FUDR) was purchased from Sigma ChemicalCompany (St. Louis, MO).  Measurement of internal manganese levels using ICP-MS (inductively coupled plasma mass spectrometry) Typically, ICP measurements were carried out in duplicatesand 2 – 3 independent trials were performed. Exactly 40 wormswere picked on Day 7 of their adult life from NGM + FUDR  plates containing no added Mn or 0.1 – 1 mM Mn(II) into amicrocentrifuge tube containing 150  μ l deionized water. Thistube was then incubated for 2 h at 98°C in a heating block todehydrate the worms and remove the added water. After dehy-dration followed by cooling, worms were digested in a heating block at 98°C for 18 h in 1 ml of 20% nitric acid (OPTIMA Nitric Acid, Fisher Scientific). A blank containing no wormswas also prepared each time to assess the background levels of metal ions introduced by this procedure. The digested sampleswere diluted to 3.6 ml in nanopure water and the metal levelswere measured using a Hewlett Packard (HP)-4500 ICP/MS(with an auto sampler) at California Institute of Technology(Pasadena, CA). A standard curve was generated each timeusing a series of metal ion solutions of known concentration 1186  Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185  –  1193  (typically 10 – 200 ppb). Thus, the unknown concentration of the metal ions in the worm digest sample could be determined based on the standard curve and the background metal level (inthe blank).  Monitoring development of the worms in the presence of  exogenous Mn(II) In order to understand how the worms handle manganeseion supplementation, development of wild type  C. elegans  wasmonitored from eggs to adults on NGM plates containingvarying amounts of added manganese. The concentrations of MnSO 4  tested were 0 – 2.0 mM. About 10 eggs per plate wereobtained by incubating 2 to 3 egg-laying (gravid) worms oneach plate for 2 h, after which the adult worms were discardedfrom the plates. These plates were incubated at 20 ± 1°C andmonitored every 12 h until new eggs were seen. For eachcondition 10 worms were used and the experiment was repeated4 times.  Fertility measurements Worms were grown to L4 stage starting from eggs obtainedthrough a standard egg-laying procedure as noted above. L4worms were placed individually onto the OP50 lawn onseparate NGM plates with or without added Mn(II). Duringthe egg-laying period, worms were moved twice daily to new plates and then eggs and freshly hatched L1s were counted asegg count. Plates were retained and when the worms reachedthe L4 stage they were picked and counted to obtain L4 count (to verify the egg count number). In each trial, at least 6individuals per strain per condition were used and the exper-iment was repeated 2 – 4 times.  Longitudinal automated thermotolerance assay Thermotolerance was measured using a Fluoroskan Ascent fluorometer (Thermo Labsystems, MA) following the method-ology previously described [21]. Briefly, animals were dis- pensed into a 384-well microtiter plate with each wellcontaining S medium,  E. coli  OP50 at a concentration of 5 × 10 7 cells/ml, 5  μ g/ml of cholesterol, and 1  μ M SYTOXgreen. The sealed plate was then placed into the fluorometer and the temperature set to 35°C. Fluorescence was measured ineach well every 30 min over a 20- to 24-h period, with a 20-msintegration time for each well. For SYTOX green fluorescence,the excitation wavelength was set to 485 nm and the emissionwavelength 538 nm. Differences in thermotolerance wereassessed using the Mantel – Haenszel log-rank test as implemen-ted in Prism (GraphPad Software Inc.). Kaplan – Meier survivalcurves were generated by using Prism survival analysis.  Life span measurements Life span measurements were performed using well-estab-lished methods. Synchronous worms were obtained from alarge-scale isolation of eggs from an egg-laying procedureand grown on NGM plates with OP50. When the larvae reachedL4 stage, 20 worms were transferred onto individual OP50-spotted plates with or without added ionic manganese (0, 0.1,0.5, and 1.0 mM MnSO 4 ), and this was recorded as  “ Day zero ” of the experiment. The next day,  “ Day 1 ”  of the life spanexperiment, all of the worms were inspected and any abnormalworms were removed from the experiment. When the wormsreached the gravid stage, they were transferred each day ontonew plates to avoid confusing parents with progeny. After thegravid stage, worms were transferred to new plates every 5 daysto ensure that the ionic manganese levels remained constant andthat there was plenty of food on the plates. Only the animalsthat died of natural death were processed and analyzed, whileall deaths considered unnatural (internal progeny, burrowed,lost, etc.) were censored. For each condition, there were at least 100 worms per independent trial at the beginning of anexperiment and the experiment was repeated 2 – 5 times.In order to prevent the appearance of progeny during the lifespan and ICP-MS experiments, 40  μ M 5-fluoro-2-deoxyuridinewas used in NGM plates along with or without the added Mn(II). NGM plates containing ionic manganese and FUDR were prepared in the same manner as unsupplemented NGM plates.For life span determination on plates containing FUDR, asimilar procedure as in the previous life span experiment wasfollowed except that the worms were only transferred to new plates every 5 days throughout the entire experiment. Statistical analysis Survival data obtained from the life span measurementswere subjected to Kaplan-Meier Survival curve analysis usinga commercial statistical program, StatView. A log-rank test was performed comparing untreated samples with Mn-treated sam- ples.  p  values from individual trials were used in an overallcomparison between treated and untreated by Fisher  ’ s methodfor combining probabilities. A  p  value of less than 0.05 wasconsidered to be statistically significant. Results  Mn accumulation in both wild type and mev-1 worms asdetermined by ICP-MS  We sought to determine if manganese provided in NGM plates was entering the worms and also how much manganesewas present inside the worms during subsequent life spanstudies. We picked worms from synchronous populations onDay 7 of their adult life and used ICP-MS to measure themanganese content in the worms that were grown with or without the addition of MnSO 4  (Fig. 1).Interestingly, when no extra manganese was added to the NGM plates, we could detect about 1 pmol Mn per worm. Thisamount increased significantly as we measured worms grownon plates containing Mn. We saw an increase in the internalworm Mn levels in both the wild type and  mev-1  strains withthe increased supplementation of Mn to the NGM plates. How-ever, the  mev-1  strain accumulated lower internal Mn levels at 1 1187 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185  –  1193  mM Mn(II) supplementation compared to the wild type worms.Further studies are needed to understand if the Mn uptake at 1mM is different in  mev-1  compared to the wild type worms.  Addition of 1 mM Mn(II) to the NGM plates accelerates thedevelopment of wild type worms In order to understand how wild type worms develop fromegg to adult in the presence of exogenous Mn(II), we monitoredthe development on NGM plates with and without Mn supple-mentation. We started with eggs on normal NGM plates and plates containing varying amounts of manganese (0.1, 0.5, 1.0,and 2.0 mM MnSO 4 ). The developmental stage of each indi-vidual worm was recorded at time points throughout develop-ment and a developmental rate was calculated from the overalltrend for a given condition. This developmental rate was thenused to estimate the exact time each individual animal reachedadulthood. From Fig. 2 it is clear that worms on plates contain-ing 1 and 2 mM Mn reached adulthood sooner than worms on NGM plates that did not have added Mn(II). This acceleratedrate of development is obvious at 70 h when new eggs wereseen on plates containing 1 or 2 mM Mn whereas, platescontaining up to 0.5 mM did not have any eggs at that time point, suggesting that addition of 1 mM or higher Mn(II)accelerates development of wild type worms.  Exogenous Mn 2+ at certain concentrations increaseshermaphrodite self-fertility of wild type and mev-1 worms Based on the development studies it became clear that upto 0.5 mM Mn(II) supplementation to the growth medium didnot alter the development of wild type worms. We then choseto examine the effect of Mn(II) addition on fertility. In thisstudy, along with wild type, we also used  mev-1  as this strainis documented to have reduced fertility in addition to adecreased life span. Since we wanted to use the  mev-1  strainin life span studies, we decided to test if manganese has anynegative effect on the fertility of this strain. Brood size wasdetermined according to standard protocols (Fig. 3). Theaverage wild type fertility value we obtained was 300,which agrees well with previously published measurements[22]. We were also able to observe the previously reporteddecrease in fertility for the  mev-1  strain in comparison to thewild type under normal growth conditions. Previouslyreported brood size for the  mev-1  strain was 77 and our average value was 120 [23]. The brood size of untreated mev-1  was comparable to that of   mev-1  treated with 0.5 mMMn(II). However, in the presence of 1 mM Mn(II), brood sizeincreased significantly (166 ± 31.6;  p  value determined usinga two-tailed  t   test assuming equal variance was 0.002), sug-gesting that even at 1 mM, manganese is not toxic to the  mev- Fig. 1. Internal manganese concentration determination using ICP-MS. InternalMn levels were determined using 7-day-old adults for wild type (black circles)and  mev-1  (gray triangles) grown in the presence of FUDR and varyingamounts of Mn(II) supplemented from L4 growth stage similar to the survivalstudies. Number of N2 worms analyzed for manganese content was 240 (0 – 0.5mM Mn) and 40 (1 mM Mn). For   mev-1 , total numbers of worms used in ICPmeasurements were 200 (0 Mn), 160 (0.1 and 0.5 mM Mn), and 40 (1 mM Mn).Average values with standard deviations are shown.Fig. 2. Effect of Mn(II) supplementation on the development of wild typeworms from egg to adulthood as a function of time at 20°C. Average time toadulthood ± SD is shown for each Mn condition from 4 independent experi-ments totaling approximately 40 worms per condition.  p  values were deter-mined using a two-tailed  t   test (assuming equal variance) for wild type wormswith Mn(II) treatment. Supplementation with 1 and 2 mM Mn gave statisticallysignificant differences in time to adulthood from untreated controls (1 mM,  p  = 0.014; 2 mM,  p  = 0.008).Fig. 3. Effect of Mn(II) supplementation on the total fertility of wild type and mev-1  worms at 22°C. Brood size shown in the presence of 0 (white bars), 0.5(gray bars), and 1 mM added Mn(II) (black bars) is the average ± SD from 2 to 4independent experiments. For brood size determination with 0, 0.5, and 1.0 mMMn, for wild type worm broods totaling 42, 25, and 19 were used, respectively,and for   mev-1 , 47, 35, and 12, respectively.  p  value was determined using a two-tailed  t   test (assuming equal variance). Supplementation with 0.5 mM Mn(II) for wild type and 1 mM for   mev-1  gave statistically significant differences (for wt + 0.5 mM Mn(II),  p  b  0.0001 and for   mev-1  + 1 mM Mn(II),  p  = 0.0017).1188  Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185  –  1193  1  worms. In the case of the wild type with the addition of 0.5mM Mn(II), instead of seeing any negative effect on the brood size we saw a 16% increase that was statisticallysignificant (  p  value  b 0.0001). We next tested if 1 mM Mn(II) supplementation would decrease the brood size and wesaw no significant change in wild type brood size. We saw100% egg hatching in both strains with and without Mnsupplementation under all our experimental conditions. Also,the length of the fertile period did not change significantlywith Mn(II) supplementation in both strains (data not shown).  Life span of wild type worms is not decreased by Mn(II)treatment  Since life span correlates inversely with in vivo free radicallevels and damage to biomolecules, we carried out life spananalysis using worms grown on NGM plates versus NGM plates supplemented with Mn, in order to see if Mn has any pro or antioxidant properties. Worms at the L4 stage, obtained by growing eggs on normal NGM plates, were moved to NGM plates containing no added Mn(II) or with added Mn(II) at thelevels previously noted. With the addition of up to 0.5 mM Mn(II) we noted that the wild type animals lived as long as the wildtype untreated controls (Fig. 4A). We also saw no significant decrease in the survival on addition of up to 1 mM Mn(II) in thecase of the wild type strain, suggesting that Mn is not toxic tothe worms.  Mn 2+ increases the mean life span but not the maximum life span of mev-1 worms Unlike in the case of wild type, we observed that   mev-1 worms grown from L4 with increasing Mn(II) levels reproduc-ibly had right shifted survival curves compared to untreated mev-1  worms grown under standard conditions (Fig. 4B). Al-though we do not see a maximum life span extension, theincrease in mean life span seen here is statistically significant at all Mn(II) concentrations tested. Even though the mean lifespan of   mev-1  worms grown from L4 stage on plates containingMn(II) is extended they are still significantly short-lived com- pared to wild type untreated worms.Since the use of FUDR is prevalent in life span measure-ments, we wanted to see if the increase in mean life span weobserved in the  mev-1  strain in the presence of Mn(II) was stillapparent in the presence of FUDR. It is interesting to note that with FUDR, wild type also showed a mean life span extensionwhen the Mn concentration was greater than 0.1 mM (Fig. 4C).In the case of   mev-1  with FUDR treatment, Mn is effective in Fig. 4. Effects of Mn(II) supplementation on the adult life span of wild type and  mev-1 C. elegans  hermaphrodites in the presence and absence of FUDR at 22°C. Eachexperiment was repeated 2 – 5 independent times and log-rank tests were performed comparing untreated samples with Mn-treated samples within the same strain andexperimental condition (±FUDR).  p  values from individual trials were then used in an overall comparison between treated and untreated by Fisher  ’ s method for combining probabilities and this is the value that is reported. A  p  value of less than 0.05 was considered to be statistically significant. (A) Survival curves show theeffects of Mn(II) supplementation from L4 stage on the adult life span of WT. Numbers of deaths scored were 425 (0 Mn), 386 (0.1 mM Mn), 415 (0.5 mM Mn), and184 (1 mM Mn). Only 0.5 mM Mn supplementation showed a significant difference (  p  value = 0.0265) from untreated WT control. (B) Survival curves show theeffects of Mn(II) supplementation on the adult life span of   mev-1 . Numbers of deaths scored were 258 (0 Mn), 298 (0.1 mM Mn), 278 (0.5 mM Mn), and 286 (1 mMMn).  p  values for 0.1, 0.5, and 1 mM Mn(II) supplemented were  b 0.0001, 0.0037, and  b 0.0001, respectively. (C) Life span curves show the effects of Mn(II)supplementation on WT strain in the presence of FUDR. Numbers of deaths scored were 213 (0 Mn), 209 (0.1 mM Mn), 215 (0.5 mM Mn), and 203 (1 mM Mn).Only 0.5 and 1 mM Mn supplementation gave a statistically significant difference as indicated by  p  values of  b 0.0001, and 0.0007, respectively. (D) Shows the effectsof Mn(II) supplementation on  mev-1  survival in the presence of FUDR. Numbers of deaths scored were 184 (0 Mn), 195 (0.1 mM Mn), 168 (0.5 mM Mn), and 154(1 mM Mn). Only 0.5 and 1 mM Mn supplementation gave a statistically significant difference as indicated by  p  values of 0.0163, and 0.0011, respectively.1189 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185  –  1193
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