Free Radical Biology & Medicine 40 (2006) 1185 – 1193

www.elsevier.com/locate/freeradbiomed

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 2005
Available 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 radical
scavenger in unicellular organisms, while there have been other reports that manganese causes Parkinson’s disease-like syndrome, DNA
fragmentation, 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 using
inductively coupled plasma mass spectrometry (ICP-MS) and the data obtained suggest that manganese supplemented to the growth medium is
taken 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 to
do 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 oxidation state being the most predominant in biological systems.

Manganese (Mn) is an essential ultratrace element similar to
chromium, molybdenum, and cobalt. It is needed for a wide variety
of physiological processes ranging from the regulation of repro-
duction to normal brain function. It is also required for several
enzymes including arginase, pyruvate carboxylase, glycosyl trans-
ferase, E. coli aminopeptidase, and the mitochondrial antioxidant
enzyme, manganese superoxide dismutase (MnSOD). Mn can
exist in various oxidation states ranging from −3 to +7, with +2
Abbreviations: ROS, reactive oxygen species; SOD, superoxide dismutase;
MnSOD, manganese-containing SOD; CuZnSOD, copper, zinc-containing
SOD; ICP-MS, inductively coupled plasma mass spectrometry; Mn, ionic
manganese; FUDR, 5-fluoro-2-deoxyuridine; WT (or N2), wild type worm.
⁎ Corresponding author. Fax: +1 714 278 5316.
E-mail address: chandra@fullerton.edu (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
Unlike the well-studied transition metal ions, copper, iron, and
zinc, not much is known about ionic manganese in vivo, although
several 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 growth
defects 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 levels
are high (30 mM), and it is thought that manganese complexed
to intracellular high molecular weight polyphosphate might
provide a defense against superoxide [3]. In bacterial systems,
low molecular weight manganese complexes are thought to be
working in conjunction with antioxidant enzymes such as SOD
and catalase to provide a defense against oxygen radicals [2].
Comparison of several lactic acid bacterial species indicates that
1186 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185–1193
when an organism lacks detectable SOD activity they require
higher levels of manganese than organisms that have SOD
activity [4]. A recent study showed that Deinococcus radio-
durans that are highly resistant to ionizing gamma radiation
have 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 knockouts
in media containing millimolar levels of manganese rescues all
phenotypes of the compromised knockout strain [6] even in the
absence of MnSOD, suggesting that the manganese rescue is
also independent of MnSOD activity in yeast. While redox-
active metals such as Fe(II) can accelerate lipid peroxidation,
ionic manganese (10–100 μM) has been shown to inhibit lipid
peroxidation in rat liver microsomes [7]. Also, several known
manganese complexes including the manganese salen and man-
ganese bis(cyclohexylpyridine)-substituted macro cyclic ligand
have shown promise as possible SOD mimics [8–10]. Studies
have shown that these complexes are as effective as SOD
enzymes in detoxifying superoxide under some experimental
conditions [11]. Some of these complexes also have marginal
catalase activity in addition to their SOD-like activity [9].
Despite several reports suggesting the beneficial effects of
manganese in unicellular organisms, it is well known that chronic
exposure 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 it is unclear as to whether this is provoked by apoptosis
or necrosis. Although Mn-treated cells have activated classical
apoptosis 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 manganese
has not been shown to participate in Fenton-type reactions that
lead to the generation of hydroxyl radicals, it is nevertheless
intriguing to see that in some unicellular organisms, ionic man-
ganese can compensate for lack of cellular antioxidant defenses
or provide protection against ROS-producing agents. Since all
of 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 in
a multicellular organism. C. elegans provide a valuable system
to address the pro or the antioxidant role for ionic manganese as
a number of genetic mutants with altered levels of oxidative
stress resistance are available. mev-1 encodes a subunit of suc-
cinate-coenzyme Q oxidoreductase in complex II of the electron
transport 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 elevated
oxidative stress such as sensitivity to the superoxide generator
paraquat or high oxygen [17]. The availability of this well-
characterized strain provides a useful tool for testing compounds
for antioxidant properties [18–20].
To uncover if manganese has a pro or antioxidant property in
vivo we provided varying levels of manganese to the growth
medium to see how manganese supplementation affects C.
elegans development, fertility, thermal stress resistance, and
life span. Our results indicate that ionic manganese is clearly
not toxic to the worms (up to 1 mM) as it does not decrease the
life span or the fertility of the worms. In our study we also saw
an increase in mean life span of a manganese supplemented
short-lived mev-1 strain, and under some experimental condi-
tions in the wild type strain also. In addition, we saw an
enhanced 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 growth
C. 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 culture
conditions and techniques were used. The OP50 strain used
in this study was obtained from Dr. Catherine Clarke’s Lab-
oratory (UCLA). Manganese sulfate (MnSO4) was the source
of 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 room
temperature 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 Chemical
Company (St. Louis, MO).
Measurement of internal manganese levels using ICP-MS
(inductively coupled plasma mass spectrometry)
Typically, ICP measurements were carried out in duplicates
and 2–3 independent trials were performed. Exactly 40 worms
were picked on Day 7 of their adult life from NGM + FUDR
plates containing no added Mn or 0.1–1 mM Mn(II) into a
microcentrifuge tube containing 150 μl deionized water. This
tube was then incubated for 2 h at 98°C in a heating block to
dehydrate 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 worms
was also prepared each time to assess the background levels of
metal ions introduced by this procedure. The digested samples
were diluted to 3.6 ml in nanopure water and the metal levels
were 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 time
using a series of metal ion solutions of known concentration
 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 (in
the blank).
Monitoring development of the worms in the presence of
exogenous Mn(II)
In order to understand how the worms handle manganese
ion supplementation, development of wild type C. elegans was
monitored from eggs to adults on NGM plates containing
varying amounts of added manganese. The concentrations of
MnSO4 tested were 0–2.0 mM. About 10 eggs per plate were
obtained by incubating 2 to 3 egg-laying (gravid) worms on
each plate for 2 h, after which the adult worms were discarded
from the plates. These plates were incubated at 20 ± 1°C and
monitored every 12 h until new eggs were seen. For each
condition 10 worms were used and the experiment was repeated
4 times.
Fertility measurements
Worms were grown to L4 stage starting from eggs obtained
through a standard egg-laying procedure as noted above. L4
worms were placed individually onto the OP50 lawn on
separate NGM plates with or without added Mn(II). During
the egg-laying period, worms were moved twice daily to new
plates and then eggs and freshly hatched L1s were counted as
egg count. Plates were retained and when the worms reached
the L4 stage they were picked and counted to obtain L4 count
(to verify the egg count number). In each trial, at least 6
individuals 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 well
containing S medium, E. coli OP50 at a concentration of
5 × 107 cells/ml, 5 μg/ml of cholesterol, and 1 μM SYTOX
green. The sealed plate was then placed into the fluorometer
and the temperature set to 35°C. Fluorescence was measured in
each well every 30 min over a 20- to 24-h period, with a 20-ms
integration time for each well. For SYTOX green fluorescence,
the excitation wavelength was set to 485 nm and the emission
wavelength 538 nm. Differences in thermotolerance were
assessed using the Mantel–Haenszel log-rank test as implemen-
ted in Prism (GraphPad Software Inc.). Kaplan–Meier survival
curves 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 a
large-scale isolation of eggs from an egg-laying procedure
1187
and grown on NGM plates with OP50. When the larvae reached
L4 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 MnSO4), and this was recorded as “Day zero”
of the experiment. The next day, “Day 1” of the life span
experiment, all of the worms were inspected and any abnormal
worms were removed from the experiment. When the worms
reached the gravid stage, they were transferred each day onto
new plates to avoid confusing parents with progeny. After the
gravid stage, worms were transferred to new plates every 5 days
to ensure that the ionic manganese levels remained constant and
that there was plenty of food on the plates. Only the animals
that died of natural death were processed and analyzed, while
all 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 an
experiment and the experiment was repeated 2–5 times.
In order to prevent the appearance of progeny during the life
span and ICP-MS experiments, 40 μM 5-fluoro-2-deoxyuridine
was 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, a
similar procedure as in the previous life span experiment was
followed 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 measurements
were subjected to Kaplan-Meier Survival curve analysis using
a 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 overall
comparison between treated and untreated by Fisher’s method
for combining probabilities. A p value of less than 0.05 was
considered to be statistically significant.
Results
Mn accumulation in both wild type and mev-1 worms as
determined by ICP-MS
We sought to determine if manganese provided in NGM
plates was entering the worms and also how much manganese
was present inside the worms during subsequent life span
studies. We picked worms from synchronous populations on
Day 7 of their adult life and used ICP-MS to measure the
manganese content in the worms that were grown with or
without the addition of MnSO4 (Fig. 1).
Interestingly, when no extra manganese was added to the
NGM plates, we could detect about 1 pmol Mn per worm. This
amount increased significantly as we measured worms grown
on plates containing Mn. We saw an increase in the internal
worm Mn levels in both the wild type and mev-1 strains with
the increased supplementation of Mn to the NGM plates. How-
ever, the mev-1 strain accumulated lower internal Mn levels at 1
1188 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185–1193
Fig. 1. Internal manganese concentration determination using ICP-MS. Internal
Mn 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 varying
amounts of Mn(II) supplemented from L4 growth stage similar to the survival
studies. Number of N2 worms analyzed for manganese content was 240 (0–0.5
mM Mn) and 40 (1 mM Mn). For mev-1, total numbers of worms used in ICP
measurements were 200 (0 Mn), 160 (0.1 and 0.5 mM Mn), and 40 (1 mM Mn).
Average values with standard deviations are shown.
mM Mn(II) supplementation compared to the wild type worms.
Further studies are needed to understand if the Mn uptake at 1
mM is different in mev-1 compared to the wild type worms.
Addition of 1 mM Mn(II) to the NGM plates accelerates the
development of wild type worms
In order to understand how wild type worms develop from
egg to adult in the presence of exogenous Mn(II), we monitored
the 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 MnSO4). The developmental stage of each indi-
vidual worm was recorded at time points throughout develop-
ment and a developmental rate was calculated from the overall
trend for a given condition. This developmental rate was then
used to estimate the exact time each individual animal reached
adulthood. From Fig. 2 it is clear that worms on plates contain-
Fig. 2. Effect of Mn(II) supplementation on the development of wild type
worms from egg to adulthood as a function of time at 20°C. Average time to
adulthood ± 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 worms
with Mn(II) treatment. Supplementation with 1 and 2 mM Mn gave statistically
significant differences in time to adulthood from untreated controls (1 mM,
p = 0.014; 2 mM, p = 0.008).
ing 1 and 2 mM Mn reached adulthood sooner than worms on
NGM plates that did not have added Mn(II). This accelerated
rate of development is obvious at 70 h when new eggs were
seen on plates containing 1 or 2 mM Mn whereas, plates
containing 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 Mn2+ at certain concentrations increases
hermaphrodite self-fertility of wild type and mev-1 worms
Based on the development studies it became clear that up
to 0.5 mM Mn(II) supplementation to the growth medium did
not alter the development of wild type worms. We then chose
to examine the effect of Mn(II) addition on fertility. In this
study, along with wild type, we also used mev-1 as this strain
is documented to have reduced fertility in addition to a
decreased life span. Since we wanted to use the mev-1 strain
in life span studies, we decided to test if manganese has any
negative effect on the fertility of this strain. Brood size was
determined according to standard protocols (Fig. 3). The
average wild type fertility value we obtained was 300,
which agrees well with previously published measurements
[22]. We were also able to observe the previously reported
decrease in fertility for the mev-1 strain in comparison to the
wild type under normal growth conditions. Previously
reported 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 mM
Mn(II). However, in the presence of 1 mM Mn(II), brood size
increased significantly (166 ± 31.6; p value determined using
a 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. 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 4
independent experiments. For brood size determination with 0, 0.5, and 1.0 mM
Mn, 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).
 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185–1193 1189

1 worms. In the case of the wild type with the addition of 0.5
mM Mn(II), instead of seeing any negative effect on the
brood size we saw a 16% increase that was statistically
significant (p value b0.0001). We next tested if 1 mM Mn
(II) supplementation would decrease the brood size and we
saw no significant change in wild type brood size. We saw
100% egg hatching in both strains with and without Mn
supplementation under all our experimental conditions. Also,
the length of the fertile period did not change significantly
with 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 radical
levels and damage to biomolecules, we carried out life span
analysis 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 the
levels 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 wild
type untreated controls (Fig. 4A). We also saw no significant
decrease in the survival on addition of up to 1 mM Mn(II) in the
case of the wild type strain, suggesting that Mn is not toxic to
the worms.
Mn2+ 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, the
increase in mean life span seen here is statistically significant
at all Mn(II) concentrations tested. Even though the mean life
span of mev-1 worms grown from L4 stage on plates containing
Mn(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 we
observed in the mev-1 strain in the presence of Mn(II) was still
apparent in the presence of FUDR. It is interesting to note that
with FUDR, wild type also showed a mean life span extension
when 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. Each
experiment was repeated 2–5 independent times and log-rank tests were performed comparing untreated samples with Mn-treated samples within the same strain and
experimental 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 the
effects 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), and
184 (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 the
effects 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 mM
Mn). p values for 0.1, 0.5, and 1 mM Mn(II) supplemented were b0.0001, 0.0037, and b0.0001, 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 b0.0001, and 0.0007, respectively. (D) Shows the effects
of 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.
1190 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185–1193

increasing the mean life span but at a higher concentration Table 1

(N0.1 mM) compared to that previously seen in the absence of Effect of Mn(II) supplementation on wild type C. elegans stress resistance
FUDR (Fig. 4D). Again in the case of FUDR trials with Mn(II) [Mn(II)] Median survival N p⁎
supplementation no increase in maximum life span was mM at 35°C (hr)
observed. Trial 1
0.0 9.5 45 –
0.1 9 48 0.21
Mn(II) treatment confers thermotolerance on wild type
0.5 9 46 0.47
hermaphrodites 1.0 9 45 0.39

We examined the effect of Mn(II) on survival during a Trial 2

thermal stress. Heat shock results in reactive oxygen species 0.0 10 88 –
0.1 10.5 94 0.0118
production and accumulation of oxidative damage in C. elegans
0.5 10.5 96 0.0151
(J.N. Sampayo and G.J. Lithgow, unpublished data). We ap- 1.0 11 94 b0.0001
plied an automated assay system [21] to undertake multiple
trials over three Mn(II) concentrations (Fig. 5 and Table 1). Trial 3
0.0 10 89 –
0.1 12 93 b0.0001
0.5 10.5 91 0.15
1.0 10 93 0.05

Trial 4
0.0 9.5 87 –
0.1 11 96 0.0037
0.5 9.5 95 0.59
1.0 9 95 0.0459

Trial 5
0.0 13 90 –
0.5 13.5 91 0.0397
1.0 14 89 0.0005

Trial 6
0.0 10.5 86 –
0.1 12 75 b0.0001
0.5 14 72 b0.0001
1.0 14 51 b0.0001

All combined
0.0 – –
0.1 5 trials (10 df) b0.0001
0.5 6 trials (12 df) b0.0001
1.0 6 trials (12 df) b0.0001
⁎ Survival curve comparisons (24 hour treatment and untreated controls) by
log rank test. All combined comparison between treated and untreated by
Fisher’s method for combining probabilities. Significance is taken when −2 Σ
ln p N X2.05 with 2k degrees of freedom, where k = the number of separate tests
and probabilities.

At 1 mM Mn(II) statistically significant thermotolerance was

Fig. 5. Effects of Mn(II) supplementation on wild type C. elegans hermaphro-
dite during 35°C heat shock. Panels A–C show representative survival curves
during a 35°C heat shock. Three-day-old adult hermaphrodites were treated
with Mn(II) or control vehicle (water) for 24 h on agar plates then dispensed at 1
worm per well into a 384 well plate. Survival of individuals at 35°C was
measured via SYTOX green fluorescent staining every 30 min for 20
h (n N 71) for each group. Each experiment was repeated 5–6 independent
times (see Table 1). Log-rank tests were performed comparing untreated sam-
ples with Mn-treated samples. p values from individual trials were then used in
an overall comparison between treated and untreated by Fisher’s method for
combining probabilities and these are the values reported. A p value of less than
0.05 was considered to be statistically significant. Numbers of deaths scored
were 485 (0 Mn), 481 (0.1 mM Mn), 491 (0.5 mM Mn), and 467 (1 mM Mn). p
values for 0.1, 0.5, and 1 mM Mn(II) supplemented were all b0.0001.
conferred in 4 of 6 trials; at 0.5 mM Mn(II) thermotolerance
was observed in 3 of 6 trials and at 0.1 mM Mn(II) thermo-
tolerance was observed in 4 of 5 trials. We conclude that while
there is considerable between-experiment variability, Mn(II)
confers enhanced survival during a thermal stress. This is
illustrated in Fig. 5 and in the overall comparison where statis-
tically significant thermotolerance was conferred by treatment
with Mn (II) at 0.1, 0.5, and 1.0 mM.
Discussion
In this study, the effect of millimolar levels of manganous
ion supplementation was explored using two strains of C.
 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185–1193
elegans, wild type and mev-1. Since Mn has been reported to
possess antioxidant properties in several unicellular organisms
[2], we used a multicellular organism to examine if Mn is
beneficial in a higher system as well, or if this in vivo beneficial
property is limited to only certain unicellular models. To un-
cover if Mn has any antioxidant properties similar to those seen
with non-peptidyl SOD mimics (EUK compounds) [18], we
looked for the effect of Mn on several phenotypes such as
development, fertility, life span, and thermotolerance.
In order to carry out supplementation studies with Mn, the
first step was to determine whether or not Mn is toxic to the
worms and, if so, at what concentrations. Manganous ions were
provided in the agar in the form of MnSO4 and although we
attempted to use concentrations higher than 1 mM, due to
solubility limitations we were unable to go higher than 1 mM
in the majority of experiments. Based on developmental studies
in wild type, Mn supplementation of up to 0.5 mM did not alter
the development of worms from egg to adult, while addition of 1
and 2 mM Mn accelerated development (Fig. 2). Although we
lack information as to why this is the case, one possibility is that
Mn is probably not at an optimal level in the NGM plates. It is
well known that deficiency of a number of ultratrace elements
including Mn leads to growth depression [24]. Hence, we be-
lieve that Mn supplementation fixes the inadequacy and hence
has a positive effect on development. On the other hand if NGM
was manganese replete, added Mn(II) would induce toxicity and
we would have observed retardation of growth, as we have
noticed with copper (data not shown). Addition of greater than
0.5 mM Cu(II) slowed the development of eggs to adult by 1
growth stage, supporting the idea that toxicity retards develop-
ment. Brood size, the length of the fertile period, and the
percentage egg hatch did not decrease with manganese supple-
mentation up to 1 mM in both strains. All these results suggest
that Mn is not toxic to the worms at the levels used in our
studies. In fact, Mn is somewhat beneficial to the wild type
worms at 0.5 mM and mev-1 worms at 1 mM as we see an
increase in brood size (Fig. 3). It is interesting to note that the
amount of Mn(II) needed to observe an increase in brood size is
different in the two strains. This is in accordance with the
thinking that if mev-1 is experiencing higher levels of oxidative
stress and Mn(II) was acting as a protective factor, we would
predict this strain to have a higher Mn requirement than the wild
type.
Oxidative stress conditions generated by high free radical
levels or compromised antioxidant defenses are some of the
many factors that are linked to a reduction in life span [25,26].
Since mev-1 has a reduced life span along with other well-
documented phenotypes that are consistent with elevated oxi-
dative stress, this strain provided us with a good system to test
the pro or the antioxidant properties of Mn along with wild type.
In our life span studies with the wild type (Fig. 4), Mn does not
appear to be beneficial to the worms except when the levels are
higher than 0.1 mM and when FUDR is present. This result is
not surprising based on studies done in yeast, where Mn is in
fact somewhat toxic to the wild type yeast, as it decreases
growth. This decrease in growth may be because there is suffi-
cient cellular antioxidants including SODs, and Mn is only a
1191
secondary defense against superoxide. When cellular antioxi-
dants are plentiful as one would expect in a wild type cell, Mn2+
might instead carry out undesired chemistry by replacing Mg2+
in the active site of certain enzymes, which could lead to the
observed toxicity in yeast. However, in the case of C. elegans,
Mn is not toxic as it does not provide reduced survival with or
without FUDR in either mev-1 or wild type worms. In the case
of mev-1, Mn is clearly beneficial as it provides a statistically
significant increase in mean life span (Fig. 4). It has been
previously reported that superoxide anion levels are higher and
SOD activity is decreased in this short-lived strain [16,23] and
hence one possibility is that the added Mn is taking the place of
SODs to neutralize the superoxide anions. However, it is unclear
why Mn is able to provide only mean life span extension
without any alteration in maximum life span. One possibility
could be that even though 1 mM Mn is added to the NGM
plates, very little might actually get in to the worms after the
manganese works its way through the bacteria (OP50). By using
different chemical forms of manganese or by using a chemically
defined medium without bacteria [27], it might be possible to
increase the in vivo dosage of manganese to see if a greater
benefit or some toxicity could be seen.
It is interesting to note that in the presence of FUDR, Mn is
beneficial to the wild type reproducibly at concentrations great-
er than 0.1 mM and also to mev-1 at concentrations greater than
0.1 mM. This result is different from what is seen in the absence
of FUDR. We believe that these differences could be due to one
or more of the following: although addition of FUDR alone
does not alter wild type life span [28,29], it is possible that it
may interfere with Mn uptake, accumulation, bioavailability,
and/or localization. Also, in our experiments when FUDR was
present worms were not moved to new plates every day during
the reproductive stage, unlike in the absence of FUDR, where
the worms were transferred each day (to prevent the progeny
from interfering with the experiment). This could have also
contributed to the observed differences in the presence of
FUDR by altering the levels of stress imposed by handling.
Though we can not conclusively demonstrate why the differ-
ences are seen, the fact remains that we do not see any negative
influence of Mn supplementation on life span.
As reported previously by Ishii et al. [20], vitamin E does not
provide an increase in mean or maximum life span in mev-1
worms. This result is consistent with the idea that vitamin E,
although an antioxidant, in general is not a good superoxide
scavenger as the rate constant for the reaction between superox-
ide and vitamin E is much slower (1000- to 10,000-fold) than
the rate constant of the reaction between superoxide and nitric
oxide [30]. It is also possible that vitamin E simply did not get to
the site of superoxide production or the concentration used was
not high enough. However, our life span results shown here
(Fig. 4) in the same strain suggest that Mn might be more
specific for scavenging superoxide. It is also important to note
that while wild type worms grown on NGM plates supplemen-
ted with iron have reduced mean and maximum life spans as
shown by Kim et al. [31], we demonstrate here that Mn(II) up to
1 mM can extend mean life span. This indicates that although Fe
and Mn are both redox-active transition metals, they might have
1192 Y.-T. Lin et al. / Free Radical Biology & Medicine 40 (2006) 1185–1193
somewhat different properties in vivo. For instance, a previous
report describes lipid peroxidation studies where Fe promoted
lipid peroxidation while Mn was found to be beneficial [7].
These results taken together suggest that Mn has some unde-
fined antioxidant property, in contrast with iron, which is clearly
a prooxidant and thus lowers life span in C. elegans.
Thermotolerance is a common characteristic of long-lived
mutants and can also be conferred by treatment with manga-
nese salen SOD mimetics [19,32] and other small molecule
antioxidants (G.J. Lithgow, unpublished data). Here we have
shown that Mn(II) alone can confer significant increases in
resistance to this environmental stress. Thermotolerance can
often result from a mild-stressful pretreatment [32] but such
pretreatments are always associated with a massive decline in
fertility [33]. Since Mn(II) has no effect on fertility at the
doses that confer thermotolerance, it is unlikely that Mn(II) is
inducing a stress.
Many studies performed with non-peptidyl synthetic SOD
mimics (EUK compounds) have not provided direct evidence
thus far to support the uptake of manganese-containing test
compounds in the worms. Dose-dependent life span data
obtained provided indirect evidence in those studies for the
intake of these compounds [18]. Here, for the first time, we
have used ICP-MS methodology to show that the worms grown
on plates containing manganese do indeed accumulate manga-
nese and we are able to calculate manganese levels on Day 7 of
their adult life (Fig. 1). The dose-dependent increase in accu-
mulation of manganese is clear evidence that Mn added to the
NGM plate is taken in by the worms. Although we were able to
calculate picomoles of Mn per worm in the two strains under
different conditions, it should be noted that the amount shown
here is on Day 7 of adult life span where the worms were
feeding on bacteria that were not removed from the gut prior
to ICP sample collection. However, it is likely that the E. coli
contribution to the overall Mn level is negligible.
The evidence presented in this paper supports the conclusion
that manganese is not toxic to the worms and it is beneficial at
the levels used in this study. Unlike the synthetic, Mn-contain-
ing SOD mimics used in C. elegans aging studies, we are able
to use a simple manganese salt (MnSO4) to provide benefit to
the worms, suggesting that it is Mn that is needed and possibly
not the counterion. Although it is likely that solubility, uptake,
localization, and bioavailability of Mn can be altered by the
counterion. It is important to note that the SOD mimics (EUK
compounds) have failed to extend life span in C. elegans under
all conditions [34]. In this study, we have used standard growth
conditions under which the worms have normal brood size,
indicating that caloric restriction is unlikely to play a role.
Although we do not see a 40% increase in life span previously
reported for mev-1 using EUK compounds under certain culture
conditions [18] that had a significantly reduced brood size,
simple manganese salt effects in comparison seem to be mar-
ginal under standard growth conditions. However, it should
also be noted that even the smallest increase of mev-1 life
span extension with Mn (11%) is the same magnitude of the
change observed in human life expectancy over the last 55
years (75 years to 84, for females in developed countries). In
other words, this is a highly significant change in survival. As
stated earlier, by determining optimal levels of Mn through the
use of altered chemical forms of manganese and/or growth
conditions, one may achieve more benefit.
To understand how manganese works in vivo in the nema-
tode, studies that measure oxidative stress and damage along
with Mn uptake are needed to fully understand the beneficial
effects of manganese. Mechanistically, it is unknown how
manganese works to scavenge superoxide radicals in vivo. We
propose that manganese might directly scavenge radicals like
an SOD and/or up-regulate several key antioxidant enzyme
defenses including manganese containing SODs to exert its
action. There is some evidence supporting this line of thought
as reduced pmr-1 expression (a P-type ATPase involved in
calcium and manganese homeostasis [35,36]) results in elevat-
ed Mn levels that suppress the paraquat (a superoxide genera-
tor) sensitivity of a daf-16 (forkhead transcription factor
involved in insulin-like signaling, stress resistance, and life
span determination in C. elegans) mutant in vivo [37]. Future
studies are needed to understand the mode of action of manga-
nese ions in oxygen radical scavenging.
Acknowledgments
This research is supported by an award from the Research
Corporation, California State University Program for Education
and Research in Biotechnology (CSUPERB), and NIH
AG024163 to C.S. G.J.L. is supported by the NIH AG21069,
AG22868, NS050789-01 and the Ellison Medical Foundation.
The COPAS BIOSORT system used in the automated survival
assay was a generous gift from The Glenn Foundation for
Medical Research and The Herbert Simon Foundation. All
nematode strains were obtained from the Caenorhabditis Ge-
netics Center, funded by the National Institutes of Health Na-
tional Center for Research Resources. The authors thank
Catherine Clarke, Oscar Aurelio, and other worm lab members
for helpful discussions.
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