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First published online November 28, 2008
Journal of Experimental Biology 211, 3816-3825 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.021303
Hypotaurine and sulfhydryl-containing antioxidants reduce H2S toxicity in erythrocytes from a marine invertebrate
University of Florida, P.O. Box 118525, Department of Zoology, Gainesville, FL 32611, USA
* Author for correspondence (e-mail: djulian{at}ufl.edu)
Accepted 16 October 2008
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Summary |
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Key words: antioxidants, hydrogen sulfide, hypotaurine, invertebrate, oxidative stress
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). In
addition to inhibiting cytochrome c oxidase
(Dorman et al., 2002;
Khan et al., 1990;
Nicholls and Kim, 1982) and a
variety of other enzymes (Bagarinao,
1992), sulfide may increase oxidative damage via the
generation of free radicals. Chen and Morris first recognized that the
oxidation of sulfide would result in a chain of redox reactions, producing a
variety of free radical intermediates, including reactive oxygen species and
sulfur-centered free radicals (Chen and
Morris, 1972). This was later confirmed experimentally in seawater
(Tapley et al., 1999) and in
animal tissues in vitro (Tapley,
1993; Truong et al.,
2006). However, sulfide may also have antioxidant properties:
H2S can directly scavenge reactive oxygen and nitrogen species
(Whiteman et al., 2004;
Whiteman et al., 2005), and
reduce lipid peroxidation in a mouse model of hepatic ischemia-reperfusion
injury (Jha et al., 2008).
Nonetheless, oxidative stress is increased in at least some marine organisms
during sulfide exposure (Abele-Oeschger and
Oeschger, 1995; Joyner-Matos
et al., 2007; Julian et al.,
2005; Tapley,
1993), as well as in mammalian cells exposed to sulfide in
vitro (Attene-Ramos et al.,
2007; Eghbal et al.,
2004). Furthermore, environmental conditions other than sulfide,
in particular high concentrations of dissolved metals, may contribute to
oxidative stress at hydrothermal vents
(Bebianno et al., 2005).
Animals in sulfidic habitats appear to minimize the toxic effects of sulfide
primarily by reducing the free sulfide concentration, especially by enzymatic
oxidation of sulfide to thiosulfate and by covalent binding of sulfide with
sulfhydryl groups of `sulfide-binding proteins'
(Arp and Childress, 1983;
Arp et al., 1984;
Arp et al., 1987;
Grieshaber and Völkel,
1998; O'Brien and Vetter,
1990).
Hypotaurine (HT), the reduced metabolic precursor of taurine, is an organic
osmolyte that is present in high concentrations (as much as 150 mmol
kg–1 tissue wet mass) within the hemolymph and cells of many
marine invertebrates at deep-sea hydrothermal vents and hydrocarbon seeps
(Alberic, 1986;
Horak et al., 2003;
Pruski et al., 2000a;
Rosenberg et al., 2006;
Yancey, 2005;
Yancey et al., 2002b;
Yin et al., 2000). HT is an
effective hydroxyl radical scavenger in mammalian cells in vitro
(Aruoma et al., 1988), and HT
and its precursors, including cysteamine, cysteinesulphinic acid and cysteic
acid, have been proposed to act as antioxidants in mammalian brain, male
reproductive tissue and liver cells (Aruoma
et al., 1988; Donnelly et al.,
2000; Fellman and Roth,
1985; Huxtable,
1992; Schurr and Rigor,
1987). Therefore, HT may reduce sulfide toxicity by scavenging
free radicals produced from sulfide oxidation. However, HT can also react with
sulfide directly, forming thiotaurine (ThT) in vitro
(Cavallini et al., 1963). If
this reaction occurs in vivo, it would scavenge free sulfide and
might, therefore, reduce sulfide toxicity by acting as a sulfide `buffer'
(Alberic and Boulegue, 1990;
Brand et al., 2007;
Joyner et al., 2003;
Pruski and Fiala-Medioni,
2003; Rosenberg et al.,
2006). In sulfide-exposed animals that lack sulfide-oxidizing
endosymbionts, the resulting ThT may be enzymatically recycled back to HT,
thereby releasing sulfide at a rate that allows detoxification through other
mechanisms (Rosenberg et al.,
2006). In sulfide-exposed animals with sulfide-oxidizing
endosymbiotic bacteria, the conversion of HT to ThT may provide a mechanism to
transport sulfide to the endosymbionts, which would then convert ThT back to
sulfide and HT (Pranal et al.,
1995; Pruski and
Fiala-Medioni, 2003; Pruski et
al., 1997; Pruski et al.,
2001; Pruski et al.,
2000b). Accordingly, Pranal and colleagues proposed that the ratio
of ThT to HT in tissues represents the extent of that animal's recent sulfide
exposure (Pranal et al.,
1995). This was confirmed by Brand and colleagues and Rosenberg
and colleagues for vent species with and without sulfide-oxidizing
endosymbionts, respectively (Brand et al.,
2007; Rosenberg et al.,
2006). Therefore, HT may have two beneficial actions in
sulfide-exposed animals: firstly, scavenging free radicals generated by the
oxidation of sulfide and secondly, scavenging sulfide by reacting with it to
form ThT (Joyner et al., 2003;
Pruski et al., 2000b;
Yancey et al., 2002b).
However, whether HT actually reduces sulfide toxicity and, if so, whether this
is primarily achieved by scavenging free radicals or by scavenging sulfide
have never been directly tested.
In the present study, we investigated whether HT, ThT, antioxidants
containing sulfhydryl groups and antioxidants without sulfhydryl groups can
reduce the cytotoxicity of sulfide exposure in vitro. Sulfide
cytotoxicity was assessed by exposing erythrocytes from the bloodworm
Glycera dibranchiata Ehlers 1868 (Annelida: Polychaeta: Phyllodocida:
Glyceridae) to sulfide for 2 h, followed by the application of vital
fluorescent dyes to determine cell viability as indicated by plasma membrane
integrity. G. dibranchiata inhabits mudflats along the North Atlantic
coast of the United States, where it is probably exposed to sulfide. It lacks
a vascular system and its coelomic, hemoglobin-containing erythrocytes are
circulated to the parapodia for gas exchange
(Mangum, 1994) and, therefore,
are at risk for sulfide exposure. Body wall and coelomocyte extracts from
G. dibranchiata have been reported to contain HT but the
concentrations were not determined (Costa
et al., 1980). However, Yancey indicated that Glycera sp.
are among the invertebrates for which HT is not a major organic osmolyte in
muscle or whole-body tissue (Yancey,
2005; Yancey et al.,
2002b). This suggests that if G. dibranchiata tissues
contain endogenous HT, the concentrations are probably comparatively low.
Consequently, an effect of exogenous HT on G. dibranchiata cell
viability would presumably be independent of any physiological adaptations
that might otherwise utilize endogenous HT.
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MATERIALS AND METHODS |
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;
Pocernich et al., 2001); (4)
N-acetylcysteine (NAC, 0.03, 0.3, 1.0 mmol l–1), a
cell-permeant N-acetyl derivative of L-cysteine that is a glutathione
precursor and is commonly used at 0.5–20 mmol l–1
in vitro (e.g. Konarkowska et
al., 2005; Yan et al.,
2006); (5) L-ascorbic acid (ASC, 0.001, 0.01, 0.1 mmol
l–1), i.e. vitamin C, which is cell-impermeant and is
commonly used at 0.001–0.1 mmol l–1 in vitro
(e.g. Yedjou et al., 2008);
(6) 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol, 0.03, 0.3, 3.0
mmol l–1), a cell-permeant nitroxide antioxidant that
scavenges superoxide radicals and is commonly used at 0.003–10 mmol
l–1 in vitro (e.g.
Samuni et al., 2004;
Singh et al., 2007); and (7)
6-hydoxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®, 0.001,
0.01, 0.1 mmol l–1 from a 100 mmol l–1 stock
solution in ethanol), a water-soluble, cell-permeant analog of
-tocopherol that is commonly used at 0.002–100 mmol
l–1 in vitro with mammalian cells (e.g.
Bhattacharya and Lakshmana Rao,
2001; Casado et al.,
2007) and mussel hemocytes
(Machella et al., 2006),
although its aqueous solubility is limited above 1.8 mmol l–1
(Wu et al., 1990). All
compounds except ThT were obtained from Sigma Chemical Co. (St Louis, MO, USA)
and were the highest quality available. ThT was prepared by reacting HT with
sulfide as described previously (Cavallini
et al., 1963; Joyner et al.,
2003).
Erythrocytes
G. dibranchiata were purchased from Harbor Bait Company,
Wiscasset, ME, USA, which collected the worms from mudflats during low tide.
The worms were maintained in the laboratory unfed in filtered, 15°C
chilled seawater for no longer than two weeks before being used. Worms were
removed from the aquarium immediately prior to experiments, and the coelomic
fluid was drained from an incision in the proboscis and then centrifuged for
18 s at 12,000x g in order to remove the supernatant and
overlying white cell layer. The pelleted erythrocytes were further purified by
centrifugation for 10 min at 170xg and 10°C into a
25% sucrose–seawater cushion. The erythrocytes were then washed with
cold incubation buffer (sterile-filtered seawater with 10 mmol
l–1 Hepes and 0.1% glucose, pH 7.6, 1000 mosmol
kg–1) to remove sucrose residue, and the pelleted
erythrocytes were then diluted 140-fold in cold incubation buffer.
H2S exposure
Sulfide exposures were conducted by exposing a thin layer of incubation
buffer, either with or without erythrocytes, to a mixture of H2S
gas in air. This was performed in five gas-tight chambers, each containing a
black-wall, clear-bottom 96-well microplate (Corning Life Sciences, Lowell,
MA, USA), in which wells contained 50 µl of incubation buffer. An
appropriate volume of H2S gas (from a compressed tank of 99% pure
H2S gas) was then added by syringe to the gas space of each chamber
to obtain 0 (control), 0.10, 0.32, 1.0 and 3.2% H2S in air. After
exposure, the sulfide concentration in each well was determined with a
Methylene Blue assay (Gilboa-Garber,
1971) modified for 50 µl sample volumes.
To determine the effect of HT and the antioxidants on the dissolved sulfide concentration, 50µl of incubation buffer containing HT (0.5, 5.0, 50 mmol l–1) or the highest concentrations of GEE, NAC, ASC, Tempol and Trolox were exposed to H2S for 2 h at 22–24°C, after which the dissolved sulfide concentration was determined, all as described above (note that ThT was not tested in this experiment due to the expense of synthesizing it, given the low probability that it would have any effect on dissolved sulfide).
To determine the effect of sulfide on cell viability, 50 µl of erythrocytes (diluted 1:1400 in incubation buffer) was added to microplate wells and exposed to H2S gas, as described above, for 2 h at 15°C. This experiment was independently replicated with erythrocytes from three worms, with erythrocyte samples from each worm being exposed to control conditions and each level of H2S (i.e. a fully balanced design). At each H2S concentration for each replicate, an additional well containing 50 µl of incubation buffer without erythrocytes was used for the determination of the dissolved sulfide concentration, as described above.
To determine whether the addition of HT, ThT or any of the antioxidants affected cell viability during sulfide exposure, erythrocytes (diluted 1:1400 in incubation buffer) were pre-incubated with HT, ThT or an antioxidant for 1 h at 15°C in the microplates, after which H2S gas was added, as described above, and the erythrocytes were incubated for an additional 2 h. Following the incubation, the cells were labeled with vital fluorescent dyes and assessed for viability, as described below. This experiment was independently replicated with erythrocytes from five worms, with erythrocyte samples from each worm being exposed to all concentrations of each compound under control conditions and at each level of H2S (i.e. a fully balanced design).
Cell viability
To assess erythrocyte viability, a vital fluorescent dye mix was created in
incubation buffer by adding calcein [loaded as calcein green
acetoxy-methylester, a cell-permeant cytoplasmic stain that labels cells with
intact plasma membranes; 10 mol l–1 from a 2 mmol
l–1 stock solution in dimethyl sulfoxide (DMSO)], propidium
iodide (PI, a cell-impermeant nucleic acid stain that labels cells with
compromised plasma membrane integrity; 5 µgml–1 from a 1
mg ml–1 stock solution in H2O) and Hoechst 33342
(a cell-permeant nuclear stain that is used to identify cells; 2
µgml–1 from a 1 mg ml–1 stock solution in
H2O). This dye mix was prepared daily from stock solutions stored
at –20°C before use. Immediately after the erythrocytes were exposed
to sulfide, as described above, 5 µl dye mix was added to each
erythrocyte-containing well in each microplate and the dyes were allowed to
load into the cells for 25 min in the dark at room temperature. After dye
loading, 100µl incubation buffer was added to each well, which diluted any
unloaded dyes to reduce background fluorescence. All dyes were from Invitrogen
Corporation (Carlsbad, CA, USA).
For fluorescence detection of the fluorescent dyes, each microplate was placed on an epifluorescence microscope (Olympus IX-70, Olympus America Inc., Center Valley, PA, USA) with a DAPI/FITC/TRITC triple bandpass polychroic emission filter set (Chroma Technology, Rockingham, VT, USA). The dyes were excited with light from a 100 W mercury bulb through appropriate excitation filters (484/15 nm for calcein, 555/15 nm for PI and 360/40 nm for Hoechst), and digital monochrome images were recorded with a cooled CCD camera (2000R, QImaging, Burnaby, BC, Canada). The images were later analyzed using ImageJ software (v. 1.37c; http://rsb.info.nih.gov/ij/). Cells were placed into three categories based on calcein and PI labeling: (1) cells that were labeled by calcein but not PI were considered viable; (2) cells that were labeled by PI with nuclear-specific staining were considered non-viable; and (3) cells that were labeled by PI with cytoplasmic staining were considered transitional and, although probably lethally injured, were not counted in the statistical analyses. The latter category was never present in erythrocytes from control conditions. Hoechst labeled both viable and non-viable erythrocytes and was used as a nuclear marker.
Statistical analyses
All data are presented as means ±1 standard error of the mean
(s.e.m.), unless noted otherwise. The effects of HT and the antioxidants on
dissolved sulfide concentration were analyzed by two-factor analysis of
variance (ANOVA) followed by the Tukey HSD test (JMP 7.2, SAS Institute, Cary,
NC USA). In the graphs, data for PI labeling represent both cytoplasmic and
nuclear staining but statistical analysis of PI labeling was restricted to
data for nuclear staining to be maximally conservative (see Discussion). The
effects of H2S on the fractions of cells labeled by calcein and PI
were each analyzed as a split plot design, with antioxidant (including HT and
ThT) as the whole plot factor, H2S as the split plot factor and the
interaction between animal and antioxidant as the error term for the whole
plot factor. This design reduces the residual variation resulting from
between-worm differences in cell viability. Because the fractions were not
normally distributed (i.e. the majority of cells in any well were typically
either viable or non-viable), the data were analyzed with a generalized linear
mixed model (GLIMMIX, SAS software, SAS Institute). However, this analysis
could not be performed for treatments that had zero variance (i.e. when all
cells in a treatment were calcein-positive, calcein-negative, PI-positive or
PI-negative) and, therefore, tests of statistical significance were limited to
0.10% H2S for calcein and 0.32% H2S for PI, which were
selected because they are closest to the LD50 for each dye (see
Results).
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RESULTS |
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In contrast to the effect of HT, adding increasing concentrations of ThT generally decreased labeling by calcein and increased labeling by PI in cells exposed to H2S (Fig. 3, ThT panels A–D). Adding ThT at 0.50 and 5.0 mmol l–1 significantly decreased the viability of cells exposed to H2S, based on both calcein labeling (Fig. 3, ThT panel E; Table 1) (F2,5=49.9, P<0.0005, N=3) and PI labeling (Fig. 3, ThT panel F; Table 1) (F2,5=16.0, P<0.006, N=3). For example, the addition of 5 mmol l–1 of ThT at 0.10% H2S decreased calcein labeling from 0.95 to 0.40 whereas the addition of 5 mmol l–1 of ThT at 0.32% H2S increased nuclear PI labeling from 0.12 to 0.39.
Antioxidants
None of the antioxidants affected cell viability under control conditions,
indicating that these were not toxic on their own. Generally, adding GEE
increased labeling by calcein and decreased labeling by PI in cells exposed to
H2S (Fig. 4 GEE,
panels A–D). Adding GEE at 0.030 and 0.30 mmol l–1 but
not 3.0 mmol l–1, significantly increased the viability of
cells exposed to H2S, based on both calcein labeling
(Fig. 4, GEE panel E;
Table 1)
(F3,15=10.6, P<0.0005, N=4) and PI
labeling (Fig. 4, GEE panel F;
Table 1)
(F3,15=6.88, P<0.004, N=4).
Similarly, the addition of increasingly higher concentrations of NAC increased
labeling by calcein and decreased labeling by PI in cells exposed to
H2S (Fig. 4, NAC
panels A–D). All concentrations of NAC (0.030, 0.30, 1.0 mmol
l–1) significantly increased cell viability, based on both
calcein labeling (Fig. 4, NAC
panel E; Table 1)
(F3,13=17.4, P<0.0001, N=4) and PI
labeling (Fig. 4, NAC panel F;
Table 1)
(F3,13=43.6, P<0.0001, N=4).
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DISCUSSION |
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); in vivo exposure of G. dibranchiata worms
to sulfide for 24 h caused a dose-dependent increase in PI labeling of
erythrocytes and a decrease in cell proliferation at sulfide concentrations
above 0.5 mmol l–1 (Hance
et al., 2008); and sulfide exposure caused oxidative damage to RNA
and DNA in G. dibranchiata erythrocytes exposed to sulfide in
vitro and in erythrocytes and body wall of G. dibranchiata
exposed to sulfide in vivo
(Joyner-Matos et al., 2008).
However, following in vivo exposure, G. dibranchiata showed
no change in gross morphology or behavior at sulfide concentrations up to 1
mmol l–1, and short-term survival of the animals was not
affected at sulfide concentrations as high as 2.4 mmol l–1
(Hance et al., 2008).
Consequently, although G. dibranchiata can clearly be classified as
`sulfide-tolerant' based on survival during sulfide exposure, the erythrocytes
show evidence of cytotoxicity and oxidative damage both in vivo and
in vitro. These findings are consistent with sulfide exposure causing
increased free radical production and oxidative stress
(Abele-Oeschger and Oeschger,
1995; Attene-Ramos et al.,
2007; Chen and Morris,
1972; Eghbal et al.,
2004; Joyner-Matos et al.,
2006; Tapley,
1993; Tapley et al.,
1999; Truong et al.,
2006) and leads to the hypothesis that appropriate antioxidants
could increase the viability of cells exposed to sulfide.
To assess erythrocyte viability in the present study, we determined whether
the cells became labeled with the fluorescent dyes calcein and PI after
H2S exposure. Calcein (or other similar cell-permeant fluorophores)
and PI (or other similar cell-impermeant fluorophores) have long been used
together as `live/dead stains' in mammalian cells in vitro
(Dive et al., 1990) but these
have rarely been used together in marine invertebrates. However, hemocyte
viability has been assessed in bivalves and crustaceans using calcein alone
(Cardenas et al., 2004;
Marin et al., 2004) and PI
alone (Hegaret et al., 2003;
Lee et al., 2001;
Xue et al., 2001), and in
G. dibranchiata erythrocytes in vivo using PI alone
(Hance et al., 2008). We found
that under control conditions in vitro, 100% of G.
dibranchiata erythrocytes were labeled by calcein but were not labeled by
PI whereas at 3% H2S (approximately 5 mmol l–1
dissolved sulfide), 99% of the cells were labeled by PI but were not labeled
by calcein. Cytoplasmic PI labeling, whether alone or with nuclear labeling,
was more common at intermediate sulfide concentrations whereas PI labeling of
cells exposed to the highest H2S concentration was almost
exclusively nuclear. This is consistent with previous studies indicating that
non-nuclear PI labeling represents an intermediate membrane permeability state
(Dive et al., 1990).
Nonetheless, to be maximally conservative, statistical analyses of PI labeling
were performed with data for nuclear PI labeling, for which the association
with loss of cell viability is least ambiguous. These results are consistent
with H2S exposure compromising plasma membrane integrity, whether
directly or indirectly. A half-maximal effect of H2S on cell
viability (LD50) was seen at 0.32% H2S, which produces
approximately 0.3 mmol l–1 dissolved sulfide. Interestingly,
this is similar to the EC50 of 0.5–0.8 mmol
l–1 sulfide for erythrocyte mitochondrial depolarization
in vitro (Julian et al.,
2005) and the erythrocyte cell death threshold of 0.5 mmol
l–1 sulfide in vivo
(Hance et al., 2008). This
suggests a common mechanism of toxicity at moderate sulfide concentrations,
which has yet to be determined. Between-worm variability was evident in the
effect of sulfide on cell viability. For the statistical analyses, this
residual variation was reduced by the split plot design, in which samples of
cells from each animal were exposed to every H2S concentration (and
at every concentration of HT, ThT and the antioxidants). The source of this
inter-individual variation is unknown but such differences in the response of
cells to sulfide in vitro could produce variation in whole-animal
sulfide tolerance, which would be likely to affect features of populations
such as the distribution within a sulfidic habitat.
HT has been hypothesized to protect deep-sea animals from sulfide toxicity
by scavenging free radicals generated from sulfide oxidation and/or by
scavenging sulfide (Alberic,
1986; Alberic and Boulegue,
1990; Horak et al.,
2003; Pruski et al.,
1997; Pruski et al.,
2000b; Rosenberg et al.,
2006; Yancey,
2005; Yancey et al.,
2002b; Yin et al.,
2000) but whether HT reduces sulfide toxicity and, if so, by what
mechanism, had never been tested. We found that incubation buffer containing
HT had up to 86% less dissolved sulfide after 2 h exposure to H2S
than incubation buffer without HT, and that HT increased the viability of
G. dibranchiata erythrocytes exposed to H2S for 2 h. For
example at 1% H2S in the absence of HT, no cells were
calcein-positive, whereas 83% of cells were calcein-positive at 1%
H2S with 50 mmol l–1 HT. The effect on PI-labeling
was similar but less dramatic. Overall, 50 mmol l–1 HT
shifted the LD50 from 0.32% H2S to 1–3%
H2S, with lower HT concentrations having a smaller but still
significant effect. Considered alone, the cell viability data do not
distinguish whether the mechanism of protection is via scavenging
free radicals, scavenging sulfide or both. However, the positive effect of HT
on cell viability was roughly correlated with its negative effect on dissolved
sulfide concentration. This suggests, but does not prove, that sulfide
scavenging is the most important factor in the ability of HT to reduce sulfide
toxicity.
Because the ability of HT to reduce the dissolved sulfide concentration was
observed in cell-free incubation buffer, it was independent of enzymatic
activity. It would be valuable to test whether this effect is stronger in
tissues and, in particular, whether tissues of sulfide-adapted animals that
accumulate HT have specific enzymatic pathways that promote sulfide scavenging
by HT. Because we did not measure HT or ThT concentrations, we also do not
know the kinetics, equilibrium constant or other characteristics of the
reaction between sulfide and HT but, clearly, these would be valuable to
obtain, especially in tissues. Interestingly, although HT is assumed to be
cell-permeant in its zwitterionic form
(Alvarez and Storey, 1983), it
is typically concentrated primarily within cells, with lower concentrations in
hemolymph (Fiess et al., 2002;
Pruski et al., 2000a;
Yin et al., 2000), which
indicates that it is not distributed strictly by diffusion. It is unknown how
HT was ultimately distributed across the erythrocyte plasma membrane in our
experiments but it is reasonable to assume that HT reduced the intracellular
sulfide concentration as well as the extracellular sulfide concentration.
As the reaction product of sulfide and HT, ThT has no further ability to
bind sulfide. However, if ThT is an antioxidant
(Cavallini and Tentori, 1960)
it may still reduce sulfide toxicity by binding free radicals from sulfide
oxidation. Therefore, we anticipated that ThT would at worst have no effect on
cell viability during H2S exposure and at best might have a
moderate beneficial effect. However, while ThT did not affect the viability of
cells under control conditions (and was therefore not toxic itself), it
decreased cell viability during H2S exposure by as much as 83% (at
0.5 mmol l–1 ThT and 0.10% H2S). The mechanism by
which ThT increased H2S toxicity is unknown but any antioxidant
capacity it might have had was not sufficient to protect the cells.
Furthermore, this raises the question of whether, in cells incubated with HT
during sulfide exposure, gradual accumulation of ThT begins to reduce cell
viability. This exemplifies the importance of recycling of ThT to HT, whether
by sulfide-oxidizing endosymbionts or by specific enzymes
(Brand et al., 2007;
Rosenberg et al., 2006).
To further explore whether free radical scavenging alone could decrease the
toxicity of sulfide in G. dibranchiata erythrocytes, we tested
whether the antioxidants GEE, NAC, ASC, Tempol and Trolox could reduce the
dissolved sulfide concentration in cell-free buffer exposed to H2S
and increase the viability of cells exposed to H2S. Other than ASC,
these antioxidants are all cell-permeant. Unlike HT, none of the antioxidants
significantly decreased the dissolved sulfide concentration in incubation
buffer exposed to H2S but all concentrations of GEE and NAC
(0.03–3 and 0.03–1 mmol l–1, respectively)
increased the fraction of viable cells. This suggests that the beneficial
action of GEE and NAC was at least partially via free radical
scavenging, although an alternative explanation is that GEE and NAC reduced
free sulfide primarily intracellularly, which would not have been detected in
our experiments. GEE is converted within cells to the antioxidant reduced
glutathione (Halliwell and Gutteridge,
2007), which, upon becoming oxidized, can react with sulfide,
forming glutathione persulfide (Rohwerder
and Sand, 2003; Smith and
Gosselin, 1966). NAC reacts with reactive oxygen species to
produce cysteine (Aruoma et al.,
1988) and stimulates intracellular glutathione synthesis
(Moldeus et al., 1986) but it
also contains a sulfhydryl group. The remaining antioxidants, all of which do
not contain a sulfhydryl group, either reduced the viability of cells exposed
to H2S or had no effect. ASC readily oxidizes to dehydroascorbic
acid, reducing the oxidative loss of sulfide in vitro
(Glaister et al., 1984). All
concentrations of ASC (0.001–0.1 mmol l–1) decreased
cell viability. The superoxide dismutase-mimetic Tempol is a catalytic
scavenger of superoxide anions in vitro. Tempol at lower
concentrations (0.03–0.3 mmol l–1) had no effect on
cell viability but 3 mmol l–1 Tempol decreased cell viability
in the presence of H2S (e.g. by as much as 77% at 0.10%
H2S), consistent with a report that Tempol can be deleterious at
concentrations higher than 0.05 mmol l–1
(Lewinska et al., 2008).
Trolox, which inhibits membrane lipid peroxidation and protein carbonylation
(Halliwell and Gutteridge,
2007), had no effect on cell viability at any concentration
(0.001–0.1 mmol l–1) but it is important to note that
we used comparatively low concentrations so it remains possible that higher
concentrations would have been more effective. Interestingly, the persulfide
dithiothreitol (DTT) is an antioxidant that contains two sulfhydryl groups and
has been reported to reduce the lethality of H2S exposure in rats,
presumably by reducing the free sulfide concentration
(Reiffenstein et al., 1992).
We tested the ability of 0.05–5.0 mmol l–1 DTT to
increase cell viability during H2S exposure and observed a similar
effect to that of GEE and NAC; however, the highest DTT concentration
interfered with the loading, retention or fluorescence of the dyes through an
unknown mechanism and therefore the DTT data were excluded.
The ability of HT to reduce sulfide toxicity was much greater than that of
GEE and NAC but the maximum concentration of HT we used was 16-times and
50-times higher than that of GEE and NAC, respectively. HT is highly
biocompatible, and the HT concentrations we used were within the range of
tissue concentrations reported for many animals from hydrothermal vents,
although it is somewhat difficult to make comparisons with the literature
because the methods used to measure and express the HT concentration have
varied. For example, when measured as HT per tissue wet mass, reported HT
concentrations are 63–152 mmol kg–1 in several
tubeworms (Yin et al., 2000)
and 59–63 mmol kg–1 in two gastropods
(Rosenberg et al., 2006). When
measured as HT per tissue dry mass, reported concentrations are 7.2–112
mmol kg–1 in a large variety of bivalves and vestimentiferans
(Fiess et al., 2002;
Pruski et al., 2000a). Based
on these values, Yancey estimated cell solute HT concentrations of 20–60
mmol l–1 HT for Calyptogena sp. and 320–360
mmol l–1 HT for Riftia pachyptila
(Yancey, 2005).
Conclusion
HT, and to a lesser extent the sulfhydryl-containing antioxidants GEE and
NAC, increase the viability of G. dibranchiata erythrocytes exposed
to H2S in vitro. The data support the hypothesis that HT
can protect tissues of deep-sea, sulfide-adapted animals from sulfide
cytotoxicity and suggest that sulfide scavenging rather than free radical
scavenging is the most important mechanism of protection, although this does
not rule out free radical scavenging as an additional protective mechanism.
There are no reports of shallow water invertebrates with high HT
concentrations (Fiess et al.,
2002; Yancey,
2005; Yancey et al.,
2002a) but to our knowledge animals in shallow sulfidic habitats
have not been specifically investigated. Consequently, it is still unknown
whether HT or similar compounds may reduce sulfide toxicity in G.
dibranchiata or other invertebrates living in characteristically sulfidic
habitats such as mudflats and mangroves. Finally, it is evident that
H2S, from both exogenous and endogenous sources, has a variety of
physiological actions (Szabó,
2007) and, therefore, it would be valuable to determine the extent
to which HT or compounds with sulfhydryl groups may modulate the activity of
H2S in vertebrate tissues.
LIST OF ABBREVIATIONS
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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