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Expression of HSP70 in Mytilus californianus following exposure
to caffeine
Zoe Rodriguez del Rey •Elise F. Granek •
Bradley A. Buckley
Accepted: 14 March 2011 / Published online: 23 March 2011
Springer Science+Business Media, LLC 2011
Abstract Caffeine, a biologically active drug with many
known molecular targets, is recognized as a contaminant of
marine systems. Although the concentrations of caffeine
reported from aquatic systems are low (ng/l–lg/l), harmful
ecological effects not detected by traditional toxicity tests
could occur as a result of caffeine contamination. We used
Hsp70, a molecular biomarker of cellular stress, to inves-
tigate the sub-lethal cellular toxicity of environmentally
relevant concentrations of caffeine on the mussel Mytilus
californianus, a dominant species in the rocky intertidal
zone along the Oregon Coast. Hsp70 concentrations in the
gill and mantle tissue of mussels exposed to 0.05, 0.2, and
0.5 lg/l of caffeine for 10, 20, and 30 days were compared
to basal levels in control mussels. Hsp70 in the gill tissue
of M. californianus had an initial attenuation of the stress
protein followed by a significant up-regulation relative to
controls in all but the 0.5 lg/l treatment. Hsp70 in the
mantle tissue of mussels exposed to caffeine did not differ
from control mussels. This study provides laboratory evi-
dence that environmentally relevant concentrations of
caffeine can exert an effect on M. californianus gill tissue
at the molecular-level.
Keywords Mussel Ecophysiology Sublethal toxicity
Gill tissue Mantle tissue Heat shock protein
Introduction
Caffeine is among the most common organic contaminants
of surface waters and has been detected in streams, lakes,
estuaries, and oceans (Buerge et al. 2003). The concen-
trations of caffeine typically reported from aquatic envi-
ronments are in the low nanogram per liter range. Yet
caffeine is a drug with known physiological effects, even at
low concentrations, and is constantly released to the
aquatic environment via wastewater effluent and other
anthropogenic activities. It remains unclear whether such
low concentrations of caffeine, 1/1000000th the concen-
tration of a drip-brewed cup of coffee, have any measur-
able impact on aquatic organisms.
As the world’s most consumed non-prescription drug,
the majority of research on caffeine has been aimed at
understanding its effects on humans (Benowitz 1990). The
modes of action of caffeine include: (i) antagonizing
adenosine receptors; (ii) inhibiting phosphodiesterases; (iii)
sensitizing ryanodine-sensitive channels in the sarcoplas-
mic, and endoplasmic reticulum to activation by calcium;
and (iv) antagonizing GABA
A
receptors at the benzodiaz-
epine-positive modulatory site (Daly 2007). In humans,
cytochrome P450 is involved in the metabolism of caffeine
(Berthou et al. 1991). Although cytochrome P450 is highly
conserved, caffeine is more toxic to other organisms,
including horses, dogs, parrots, and spiders due to their
underdeveloped capacity to metabolize the drug (Pollack
et al. 2009).
Increasing concerns about the prevalence of caffeine in
the aquatic environment and the uncertainty of the effects
on aquatic organisms have fueled a few studies investi-
gating the effects of caffeine on aquatic organisms. Fraker
and Smith (2004) found that environmentally relevant
levels of some organic wastewater contaminants, including
Z. R. del Rey E. F. Granek (&)
Environmental Science and Management, Portland State
University, Portland, OR 97201, USA
e-mail: graneke@pdx.edu
B. A. Buckley
Biology, Portland State University, Portland, OR 97201, USA
123
Ecotoxicology (2011) 20:855–861
DOI 10.1007/s10646-011-0649-6
caffeine, had behavioral and physiological effects on
northern leopard frog (Rana pipiens) tadpoles. Gagne
´et al.
(2006) reported that, although caffeine was not very toxic
to trout (Oncorhynchus mykiss) hepatocytes, it produced
lipid peroxidation at a threshold concentration of 14 lM
after 48 h exposure at 15C. Moreover, in vitro incubation
of caffeine with trout microsomes increased both the rate of
oxidation of NAPDH and the lipid peroxidation in micro-
somes after a 60 min incubation at 30C, suggesting that
caffeine exposure could lead to oxidative damage at low
milligram per liter concentrations.
Other studies suggest that environmentally relevant
levels of caffeine are not a threat to aquatic organisms.
Smith and Burgett (2005) reported that environmentally
relevant concentrations of caffeine (0.6–600 lg/l) did not
affect the survivorship or activity of American toad (Bufo
americanus) tadpoles. Similarly, Quinn et al. (2008) clas-
sified caffeine as non-toxic based on acute (mortality) and
chronic (feeding behavior, attachment, and growth) toxic-
ity tests on the freshwater cnidarian Hydra attenuata.
Although caffeine exposure impaired the reproduction of
the water flea Ceriodaphnia dubia (IC50 =44 mg/l) and
inhibited the growth of the fathead minnow Pimephelas
promelas (IC50 =71 mg/l) (Moore et al. 2008), the
authors concluded that given the environmental concen-
trations reported in the literature, caffeine should pose
negligible risk for most aquatic vertebrate and invertebrate
organisms. The authors did caution that there could be
potential effects from long-term exposure to environmental
levels of caffeine.
Few studies have examined the effect of caffeine on
marine organisms. Cheney (1945) found that caffeine
affects oxygen consumption and the normal rate of cleav-
age division in the fertilized eggs of the sea urchin, Arbacia
puntulata. Nath and Rebhun (1976) reported inhibition of
mitosis in sea urchin eggs exposed to caffeine. Caffeine
induced bleaching in the tropical sea anemone Aiptasia
pulchella, ostensibly through its effect on levels of intra-
cellular protein phosphorylation (Sawyer and Muscatine
2001). Increased duration of exposure to caffeine resulted
in a significant increase in the percent of symbiotic algae
released from A. pulchella. A subsequent study on the
effect of caffeine on four species of coral endosymbionts
found that algal cultures grown in 60 mg/l caffeine
exhibited up or down regulation of a number of proteins
associated with glycolysis, photosynthesis, and the physi-
ological stress response (Pollack et al. 2009). Heatshock
proteins (HSP) were up-regulated 2 to threefold in the coral
endosymbiont Symbiodinium sp. and down-regulated up to
ninefold in Symbiodinium goreaui.
The previous studies show that caffeine can have a
deleterious effect on aquatic organisms. However, since
these studies have overwhelmingly focused on relatively
high concentrations of caffeine that are unlikely to be
found in aquatic environments, the risk from exposure to
low concentrations of caffeine, including effects from long-
term exposure and sublethal effects, remains unclear. Some
challenges exist in detecting impacts to organisms by
contaminants that are found at low concentrations in the
environment. Contaminants can cause changes at all levels
of biological organization and subtle or chronic biological
effects resulting in irreversible long-term changes could be
occurring in apparently healthy ecosystems (Hyne and
Maher 2003). These changes may not be initially detected
if the focus of ecological risk assessment is on coarse levels
of biological organization.
HSPs have been suggested as sensitive biomarkers of
the sub-lethal or subtle toxicity of pollutants (Sanders
1990; Depledge 1994; Lewis et al. 1999) because they are
involved in protecting and defending cells from environ-
mental offenses (Sanders 1990) and their induction is much
more responsive than traditional indices of contaminant
effects (Feder and Hofmann 1999). HSPs are proteins that
are synthesized in response to cellular stress that induces
denaturation of other proteins. The 70 kDa family (Hsp70)
is most highly conserved and has been most extensively
studied. Four key features of Hsp70 have driven its appli-
cation in environmental risk assessment: (1) it is highly
conserved in a wide variety of organisms from bacteria to
humans; (2) it responds to a variety of environmental
stresses, including thermal stress, heavy metal exposure,
organic pollutants, hypoxia/anoxia, salinity stress, and
exposure to ultraviolet radiation; (3) its induction is very
sensitive to environmental assaults; and (4) its expression
has been correlated to other toxicological end points.
Concerns about the effects of stress history, induction
thresholds and timing on expression of Hsp70 can be
minimized in laboratory studies that assess the effect of a
single contaminant and employ adequate controls for
comparison. Collecting organisms from the same envi-
ronment with adequate laboratory acclimation can help
ensure that test organisms, including controls, have a
similar stress history. Measuring the response of Hsp70 in
different tissues and over several time periods reduces the
risk of missing tissue-specific or time-dependent induction.
Hsp70 remains a potent and sensitive tool for investigating
the toxicity of contaminants of concern (Mukhopadhyay
et al. 2003).
This study investigated the toxicity of environmentally
relevant concentrations of caffeine on the common inter-
tidal mussel Mytilus californianus using Hsp70 as a
biomarker of the effects of this ubiquitous aquatic con-
taminant. Mussels are ideal marine species to investigate
the potential effects of contaminants because they are
stationary, widespread, easy to collect, filter feeders that
have been used extensively in toxicity studies using Hsp70
856 Z. R. del Rey et al.
123
as a biomarker of effect (Laporte 2005). The study
addressed the following questions:
•Is Hsp70 expression in M. californianus increased by
exposure to environmentally relevant concentrations of
caffeine?
•Is there a difference in the expression of Hsp70 in
different tissues of M. californianus exposed to
caffeine?
•Does the expression of Hsp70 change with the duration
of exposure to caffeine?
Methods
Study organisms and acclimation conditions
Mytilus californianus mussels were collected from a study
site in the coastal town of Yachats, Oregon (N 4418081.300
W 12406052.300). Mussels (10–12 cm) were collected from
the same area of the mid-intertidal zone on a single day in
September 2008 and transported in a chilled cooler to
Portland State University. In the laboratory, mussels were
acclimated in 10 gallon aquaria filled with 26 L of UV fil-
tered deionized water, adjusted to a salinity of 32 PSU
(Instant Ocean). The aquaria were connected to in-line
chillers (Sealine) and to an in-tank filter system. The accli-
mation temperature was 10–11C, the temperature at the
time of collection. After 3 days of acclimation the experi-
ment was started. Mussels were held under a natural light
cycle of approximately 12 h of daylight and 12 h of dark.
A pilot experiment verified that in-tank caffeine con-
centration did not decrease appreciably in seawater over a
7 day period. To test for caffeine degradation, a tank was
set up with twelve mussels and spiked with a known
concentration of caffeine. This set up was identical to the
experimental set up. After 1 week, 1 L samples of water
were removed from the tank and caffeine degradation from
initial concentration was verified using solid phase
extraction (EnviCarb, Supelco) and GC–MS analysis.
Experimental design
Mytilus californianus were exposed to one of four caffeine
concentrations ranging from 0.05 to 0.5 lg/l. These are
field relevant concentrations reported from coastal marine
systems (Siegener and Chen 2002; Weigel et al. 2004;
Peeler et al. 2006; Comeau et al. 2008). Caffeine solution
used to spike tanks at the beginning of the experiment and
after each water change, was prepared fresh using Caffeine
ReagentPluspowder (Sigma-Aldrich) dissolved in
nanopure water. Control mussels were not exposed to
caffeine. Twelve randomly selected mussels were placed in
each of the five caffeine treatments (0, 0.05, 0.1, 0.2, or
0.5 lg/l). The tank water was changed every 5 days and
spiked with caffeine using a freshly prepared stock solu-
tion. After each water change, mussels were fed 5 mL of
Instant Algae (Reed Mariculture).
Four mussels from each treatment were sacrificed after
10, 20, and 30 days. Mussels were dissected and a sample
of gill lamellae and mantle tissue from each mussel was
stored in separate cryovials. Sample tissues were immedi-
ately frozen in liquid nitrogen and stored in a -80C
freezer until tissue preparation. No test organisms died
during the experiment. Hsp70 concentrations in mussel
tissues were subsequently measured using the protocol
described by Buckley et al. (2001).
Tissue preparation
Approximately 100 mg of tissue was ground in a small
centrifuge tube using a pestle. The ground tissue was then
homogenized in a 1:1 (volume/volume) solution of 29
homogenization buffer consisting of 32 mM Tris–HCl (pH
6.8) and 2% sodium dodecyl sulfate (SDS). The homoge-
nate was heated to 100C for 5 min and then centrifuged at
12,0009gfor 10 min. The supernatant was collected into a
new centrifuge tube and the pellet discarded. The protein
content of the supernatant was determined by the Bradford
assay (Pierce, Rockford, IL, USA). The supernatant was
stored in a -80C freezer until further analysis. Gill
lamellae and mantle tissue were prepared using the same
procedure.
Electrophoresis and immunochemical assay of Hsp70
Equal amounts of protein (15 lg) from each sample were
separated via SDS-polyacrylamide gel electrophoresis on
ten lane 10% polyacrylamide gels. One lane in each gel
was loaded with Precision Plus Protein Kaleidoscope
standard (Bio-Rad) and one with a standard sample used to
calibrate protein expression within and among gels. The
standard sample was from one of the experimental mussel
samples. Following approximately 1.5 h of electrophoresis
at 150 V, the proteins were transferred to nitrocellulose
blots using wet electrophoretic transfer at 30 V overnight
(approximately 15 h).
Nitrocellulose blots were blocked in 5% non-fat dry
milk in 19PBS for 1 h. The immunodetection was per-
formed using an Hsp70 polyclonal antibody (Hsp70 (K-
20)-R, Santa Cruz Biotechnology) that reacts only with the
Hsp70 isoform. Blots were then incubated for 1.5 h in the
primary antibody diluted 1:5000 in 5% non-fat dry milk in
19PBS, followed by three 10-min washes in 19PBS with
0.1% Tween-20. Blots were then incubated for 1 h in
peroxidase-conjugated goat-anti-rabbit antibody (Thermo
Hsp70 expression following caffeine exposure 857
123
Scientific) diluted 1:5000 in 5% non-fat dry milk in 19
PBS, followed by six 5-min washes in 19PBS with 0.1%
Tween-20. Blocking and incubation in primary and sec-
ondary antibody was done under constant agitation at room
temperature.
Western blots were developed using Super Signal West
Pico Chemiluminescent Substrate (Thermo Scientific) and
exposed to film (Kodak BioMax MR-1). Scanning densi-
tometry, using ImageJ (http://rsb.info.nih.gov/ij/), was used
to determine the levels of Hsp70 expression relative to the
standard sample.
Data analysis
Differences in Hsp70 expression were analyzed with a two-
way ANOVA with caffeine concentration and exposure
duration as the fixed factors and Hsp70 concentration as the
dependent variable. Significant fixed factors were further
investigated using a one-way ANOVA and post-hoc Tukey
tests. Statistical analyses were performed using SPSS 17.0
(2008).
During the first 10 days of the experiment the chiller for
the caffeine treatment of 0.1 lg/l experienced a power
outage. When the power outage was discovered, the tem-
perature in that tank was 23C and could have been at that
temperature for a maximum of 48 h. Although, mussel
samples from that treatment were collected and analyzed
for Hsp70 expression, the data are not included since this
represented a thermal change that likely affected Hsp70
expression.
Results
The primary antibody used against Hsp70 was highly
specific producing bands only at approximately 70 kDa
(Fig. 1). Hsp70 expression in the gill tissue responded to
caffeine exposure. The response was time dependent, but
did not exhibit a linear dose–response relationship with
increasing concentration, although interaction between
dose and time was significant (Fig. 2; Table 1). Some of
the trends are not significant due to high variability in
Hsp70 concentrations within treatment. However, the gill
lamellae and mantle tissue of control mussels exhibited
basal levels of Hsp70 that were similar for the duration of
the experiment and had low variability.
Caffeine initially produced an inhibitory effect on
Hsp70 followed by a time and dose dependent recovery.
After 30 days, Hsp70 in the mussels exposed to 0.05 and
0.2 lg/l caffeine was up-regulated relative to controls. At
10 days of exposure to caffeine, mean Hsp70 levels of
mussels in all three caffeine treatments were lower than in
the control mussels. This trend was not statistically sig-
nificant because individual mussels exhibited marked var-
iability in the response of Hsp70, but some of the mussels
in each of the three treatments had very low or no detect-
able Hsp70. Similar variability in Hsp70 was not exhibited
by the control mussels during the time course of the
experiment.
Exposure to caffeine at 0.05 lg/l induced a moderate
up-regulation of Hsp70 in the gill lamellae of M. califor-
nianus after 20 days of exposure. Hsp70 levels remained
elevated after 30 days of exposure. In the 0.2 lg/l treat-
ment, the levels of Hsp70 did not exhibit an up-regulation
until 30 days of exposure. The maximum Hsp70 levels
detected were similar in the 0.05 and 0.2 lg/l treatments.
Interestingly, in the highest caffeine treatment (0.5 lg/l), a
similar increase in Hsp70 expression was not observed
during the duration of this experiment.
Fig. 1 Representative western blot depicting Hsp70 bands in gill
tissue from M. californianus
Fig. 2 Mean Hsp70 expression in the gill lamellae of M. californi-
anus exposed to three concentrations of caffeine for 10, 20, and
30 days. Error bars represent the range of Hsp70 concentrations
(n=4). Significant differences in Hsp70 concentration for exposure
duration within caffeine dose are indicated by the letter convention
(Pvalue \0.05). An asterisk indicates that the Hsp70 expression for
that exposure duration was significantly different from the control
(Pvalue \0.05)
Table 1 Summary of two-way ANOVA results for the gill tissue
Source Sum of squares df F Pvalue
Dose 2.336 3 3.104 0.039
Time 6.296 2 12.548 0.000
Dose 9time 6.140 6 4.079 0.003
858 Z. R. del Rey et al.
123
M. californianus exposed to caffeine exhibited a dif-
ferent pattern of Hsp70 expression in the gill lamellae and
the mantle tissue. After 30 days, Hsp70 expression in the
mantle tissue (Fig. 3) was unresponsive to caffeine expo-
sure (Table 2).
Discussion
Caffeine, a potent neuroactive drug, is recognized as an
ubiquitous contaminant in aquatic systems. Traditional
ecotoxicology endpoints suggest that the levels of caffeine
currently detected in aquatic systems do not pose a threat to
aquatic organisms. There remains a potential for sublethal
effects not detected by traditional endpoints and effects
from long-term exposure to low levels of caffeine.
This study demonstrates that Hsp70 in the gill tissue of
M. californianus responds to exposure at environmentally
relevant concentrations of caffeine. The response of Hsp70
in the gill tissue exhibited a complex pattern across dose
and time. Initially, caffeine appears to have an attenuating
effect on Hsp70 expression for all caffeine levels tested.
This trend, however, was not significant, likely due to the
small sample size and high variability of measured Hsp70
concentrations. High variability in Hsp70 expression in
response to contaminant exposure can mask trends and has
been reported in other studies (Staempfli et al. 2002;
Laporte 2005).
Increasing the duration of exposure resulted in up-reg-
ulations of Hsp70, at low to moderate caffeine concentra-
tions. Hsp70 was up-regulated after 20 days in the 0.05 lg/
l caffeine treatment and after 30 days in the 0.2 lg/l
treatment, but did not exhibit a similar increase in the
0.5 lg/l treatment over the 30 day duration of this exper-
iment. This type of Hsp70 response to caffeine exposure
may indicate a quenching phenomenon (Arts et al. 2004)
whereby high levels of stress limit HSP induction.
Only one previous study has investigated the response of
Hsp70 to caffeine exposure in aquatic organisms. Pollack
et al. (2009) assessed the effect of caffeine on coral algal
endosymbionts by identifying proteins sensitive to caffeine
exposure. Hsps were up-regulated two to threefold in the
coral endosymbiont Symbiodinium sp. and down-regulated
up to ninefold in Symbiodinium goreaui. However, the
concentration of caffeine used to incubate coral algal
endosymbionts (Pollack et al. 2009) falls within the range
that would be toxic if found in human blood serum.
Therefore, the effect on HSP proteins observed at this high
concentration of caffeine is not surprising since it can
potentially inhibit many cellular processes. All known
stresses, if sufficiently intense, induce HSP expression
(Feder and Hoffman 1999).
In contrast, the concentrations of caffeine used in this
study were in the ng/l to lg/l level. Due to the large
diversity of Hsp70 inducers, the cellular stress response is
thought to be triggered by different mechanisms of toxicity,
among which protein damage (e.g., misfolding) would be
the common link (Ait-Aissa et al. 2000). There is currently
no evidence that the low levels of caffeine tested in this
experiment are proteotoxic.
Prolonged oxidative stress from chronic exposure to
caffeine could result in an up-regulation of Hsp70 as the
organism attempts to cope with cellular damage. Gagne
´
et al. (2006) reported that exposure to caffeine could lead
to oxidative damage of trout hepatocytes. However, the
concentrations of caffeine at which trout hepatocytes were
incubated were in the mg/l range, 1,000 times higher than
the concentrations tested in this study. The role of oxida-
tive stress in inducing an Hsp70 response in the gill of
M. californianus exposed to low levels of caffeine could be
investigated further by verifying that oxidative stress is
occurring (e.g., quantifying lipid peroxidation).
Other mechanisms not related to proteotoxicity may also
induce an Hsp70 response. In humans, caffeine is able to
significantly block adenosine receptors at low serum con-
centrations (lmol) and this is considered the most common
mode of action of caffeine (Fredholm et al. 1999). Block-
ing of adenosine receptors by caffeine results in a loss of
Fig. 3 Relative Hsp70 expression in the mantle tissue of
M. californianus exposed to three concentrations of caffeine for 10,
20, and 30 days. Error bars represent the range of Hsp70 concen-
trations (n=4)
Table 2 Summary of the two-way ANOVA results for the mantle
tissue
Source Sum of squares df F Pvalue
Dose 0.181 3 2.308 0.930
Time 0.121 2 2.323 0.112
Dose 9time 0.056 6 0.356 0.902
Hsp70 expression following caffeine exposure 859
123
the inhibitory effect of adenosine and triggers a catechol-
aminergic response. Catecholamines have previously been
shown to up-regulate intracellular and extracellular Hsp72
in laboratory rats (Johnson et al. 2005).
Several studies have identified neuroendocrine and
nervous system functions in molluscs that are analogous to
the hypothalamic-pituitary system of vertebrates; similar
elements are at the basis of the response and triggering
(Stefano et al. 2002; Fabbri et al. 2008). For example,
adenosine receptors have been reported from the mussel
Mytilus edulis. Theophylline, which bears structural and
pharmacological similarity to caffeine, blocked the inhib-
itory effects of a potent adenosine analog on neurotrans-
mitter release in in vitro preparations from the pedal
ganglia of M. edulis (Barraco and Stefano 1990) In the
oyster Crassostrea gigas, circulating noradrenaline and
dopamine have been shown to increase in response to
physiological stress (Lacoste et al. 2001a).
Assuming that the mode of action of caffeine on aden-
osine receptors is similar between vertebrates and mol-
luscs, exposure to low levels of caffeine could stimulate the
release of catecholamines resulting in induction of Hsp70
in M. californianus. In at least one study, noradrenaline
was shown to induce the Hsp70 gene promoter in the oyster
Crassostrea gigas and abalone Haliotis tuberculata (Lac-
oste et al. 2001b). The authors postulated that the inte-
grated response to stress is related to the heat shock
response.
Cell signal transducers, such as changes in intracellular
pH, cyclic AMP, Ca2?,Na?, inositol trisphosphate, pro-
tein kinase C, and protein phosphatases, have also been
implicated in the modulation of Hsp70 expression (Kiang
and Tsokos 1998). For example, a study of U-937 human
promonocytic cells showed that treatment with the cAMP
increasing agent isoproterenol plus theophylline decreased
basal levels of Hsp70 (Vilaboa et al. 1995). Caffeine can
affect many of these cell signal transducers. Caffeine’s
effect on cell signal transducers could potentially mediate
an Hsp70 response.
While the potential mechanisms detailed above could
explain the observed up-regulation of Hsp70 in the gill
lamellae of M. californianus with caffeine exposure, they
do not explain the transient and concentration-dependent
attenuation of Hsp70 observed here. A quenching phe-
nomenon of Hsp70 has been described in some organisms
exposed to some types of contaminants. Arts et al. (2004)
proposed that the ability to inhibit HSP induction might
indicate a more toxic response than producing elevated
levels of these proteins, which indicates that the organism is
able to maintain homeostasis under environmental assault.
In discussing this phenomenon, Eckwert et al. (1997)
proposed that the dose–response curve for Hsp70 could be
divided into three sections: (1) homeostasis: a state of basal
Hsp70 expression, (2) compensation: a state of stress
accompanied by Hsp70 induction, and (3) non-compensa-
tion: a state of severe stress and pathological damage
blocking Hsp70 expression. This type of response was
observed in the rotifer Brachionus plicatillis. Hsp60 levels
of B. plicatillis exposed to crude oil in the laboratory were
only higher than control rotifers at very low concentrations
of crude oil (Wheelock et al. 1999). The model proposed
by Eckwert et al. (1997) does not account for the duration
of the stress, but the duration of stress can interact with
dose to alter the response of Hsp70.
Such a quenching phenomenon could explain the time
and concentration-dependent attenuation of Hsp70
observed in this study. In the case of the gill lamellae of
M. californianus, the attenuation of Hsp70 was transient
and concentration-dependent. Gill tissue from mussels
exposed to the lowest caffeine concentration exhibited a
faster recovery to levels equal to the control mussels. The
gill tissue from all but the highest treatment eventually
exhibited a time-dependent up-regulation of Hsp70, rela-
tive to controls. The actual mechanism(s) involved in
quenching (or attenuating) the HSP response is poorly
investigated, but may be the result of tissue damage
(Eckwert et al. 1997). This is not likely to be the case with
caffeine since the attenuation of Hsp70 in the gill lamellae
was transient.
Unlike the gill lamellae, the mantle tissue of M. cali-
fornianus did not exhibit an Hsp70 response over the
duration of this experiment. Although HSP expression is an
ubiquitous molecular mechanism for coping with stress, the
HSP response to stresses can be tissue-specific (Feder and
Hofmann 1999). Chapple et al. (1997) found tissue-specific
inducibility of Hsp70 in M. edulis despite all tissues being
exposed to the same temperatures. Compared with mantle
and adductor muscle tissues, gill tissue showed the greatest
increase in levels of Hsp70 proteins. Other tissues of
M. californianus may exhibit an Hsp70 response to caf-
feine exposure, but this remains to be tested.
Caffeine is a potent neuroactive drug that is recognized
as an ubiquitous contaminant in aquatic systems. M. cali-
fornianus exposed in the laboratory to environmentally
relevant concentrations of caffeine exhibited an Hsp70
response. Since Hsp70 affords cellular protection from
environmental assaults, both the up-regulation after pro-
longed exposure and the potential attenuation of the
response should be investigated further.
Acknowledgments This project was funded in part by a Portland
State University Faculty Enhancement Grant to E. F. Granek and B.
A. Buckley and by an Oregon Sea Grant Program Development Grant
to E. F. Granek. NOAA’s National Marine Fisheries Service and the
NOAA Portland, Oregon Office provided additional funding for
supplies. Malcolm Staudinger, Caitlyn Peake, Paul Pettus, Amanda
Kelly, Ben Prital, provided help in the field and laboratory.
860 Z. R. del Rey et al.
123
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