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Expression of HSP70 in Mytilus californianus following exposure to caffeine

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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-μg/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 investigate 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 μg/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 μg/l treatment. Hsp70 in the mantle tissue of mussels exposed to caffeine did not differ from control mussels. This study provides laboratory evidence that environmentally relevant concentrations of caffeine can exert an effect on M. californianus gill tissue at the molecular-level.
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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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... Literature about caffeine toxicity in non-target aquatic organisms is limited. As for bivalves, the sub-lethal cellular toxicity of environmentally relevant concentrations of caffeine was investigated in the mussel Mytilus californianus [51]. In that study, Hsp70 concentrations in the gill and mantle tissues of mussels exposed to 0.05, 0.2, and 0.5 µg/L of caffeine for 10, 20, and 30 days were compared to basal levels of the control mussels. ...
... After 14 days of exposure to caffeine in the range of 0.1-50 µg/L, significant detrimental effects were observed in the clam Ruditapes philippinarum, with a dose-dependent reduction in hemocyte lysosomal membrane stability [51]. Values of biomarkers of oxidative stress and biotrasformation phase I and II were significantly higher in digestive glands, mostly at the highest caffeine concentrations tested. ...
Article
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Combined effects of reduced pH, as predicted under climate change scenarios, and the most popular and widely used stimulant caffeine were assessed in hemocyte parameters of the mussel Mytilus galloprovincialis, being hemocytes involved in immune defense. Bivalves were exposed for one week to natural pH (8.1) and two reduced pH values (pH -0.4 units and pH -0.7 units). Exposure continued for additional two weeks, both in the absence and in the presence of environmentally relevant concentrations of caffeine (0.05 and 0.5 µg/L). Hemocyte parameters (total hemocyte count, hemocyte volume and diameter, neutral red uptake and hemocyte proliferation) were measured after 7 days of exposure to pH only, and after 14 (T1) and 21 (T2) days of exposure to the various pH*caffeine combinations. At all sampling times, pH significantly affected all the biological variables considered, whereas caffeine exhibited a significant influence at T2 only. Among the various hemocyte parameters, caffeine caused a significant increase in total hemocyte count at T2, and in hemocyte volume and diameter at both T1 and T2, when a significant interaction between pH and caffeine was also found. Overall, results demonstrated that hemocyte functionality was strongly influenced by the experimental conditions tested. Further studies are needed to assess combined effects of climate changes and emerging contaminants on bivalve immune system when challenged with environmental pathogens.
... The literature regarding the ecotoxicological effects of caffeine is diverse, and adverse effects were observed from ng/L to mg/L. To name a few examples, oxidative stress and cellular damage in mollusks (Del Rey et al. 2011;Aguirre-Martínez et al. 2015;Cruz et al. 2016) affect negatively microbial activity, respiration, and biomass (Bunch and Bernot 2011;Rosi-Marshall et al. 2013) and behavior and neuromuscular effects in fish (Chen et al. 2008, Steele et al. 2018. Moreover, when caffeine was mixed with other pharmaceuticals, a synergetic effect was found affecting aquatic communities' composition (Lawrence et al. 2012), and then mixture effects should be taken into account. ...
Chapter
Caffeine is one of the most consumed substances worldwide, and although it is present in many food products in different concentrations, coffee is likely the most significant product in terms of caffeine concentration and consumption. Despite being known and used by humankind for hundreds of years, the patterns of consumption and environmental risks are not fully understood. Therefore, global spatial and temporal caffeine consumption was investigated over the last 20 years, and health perspectives were discussed. An increase of 37% in per capita coffee consumption was observed in the last two decades worldwide, mainly in the Middle East and North Africa (84.2%) and upper middle-income countries (86.1%). Although there is still controversy on whether it is an addictive substance, it is worth noting that risks associated with its high consumption exist, mainly by more sensitive people and when mixing it with other substances, such as alcohol.
... Among the high-risk/high-frequency detected compounds, all three compounds exert adverse effects on aquatic life (Wang et al., 2021). First, reports related to the adverse effects of caffeine on different taxa are diverse; they include oxidative stress and cellular damage in bivalves and polychaetes (Cruz et al., 2016;Pires et al., 2016;Del Rey et al., 2011), and affectation of microbial activity, including different processes of the nitrogen cycle (Bunch and Bernot, 2011), all of these at environmental relevant concentrations of micrograms per liter. Similarly, higher caffeine concentration values have revealed chronic effects such as impaired reproduction in microcrustaceans, angiogenesis and reduced movement activity in fish embryos and growth inhibition in fish (Chen et al., 2008;Moore et al., 2008;Yeh et al., 2012). ...
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The occurrence of pharmaceuticals in surface water has been barely studied in Latin America. This work aimed to i) develop a multi-residue liquid chromatography- triple quadrupole mass spectrometry (LC–MS/MS) method for the determination of pharmaceutical active compounds (PhACs); ii) monitor 70 PhACs in three urban-influenced rivers in San José, Costa Rica; and iii) perform the risk assessment of detected compounds and ecotoxicological evaluation on water samples. Caffeine, 1,7-dimethylxanthine, naproxen, gemfibrozil and ibuprofen were the most frequent among 23 detected compounds. Concentrations ranged from 0.013 μg/L to 53.8 μg/L (62 % detections between 0.1 μg/L–1 μg/L), and the highest values corresponded to caffeine, 1,7-dimethylxanthine, ofloxacin, gemfibrozil and cephalexin. The environmental risk estimated using the hazard quotient (HQ) approach, revealed four and eleven compounds with medium and high risk, respectively. The highest risk (HQ >10) was determined for diphenhydramine, risperidone, fluoxetine, trimethoprim, ofloxacin and azithromycin; nonetheless, high risk (HQ >1) was also estimated for caffeine, diclofenac, clarithromycin, gemfibrozil and ibuprofen. Total HQ sample values (ΣHQ), calculated as the sum of individual HQs for each detected compound, revealed the highest hazard in surface water near wastewater treatment plant discharges, followed by the locations of higher urban influence (in the Virilla river). Ecotoxicological evaluation showed no acute toxicity towards Daphnia magna and Vibrio fischeri in surface water samples; on the contrary, toxicity towards Lactuca sativa (germination tests) showed a similar pattern to that determined with the ΣHQ, although the highest toxicity was observed downstream with respect to the urban influence of the metropolitan area. These findings help to visualize the importance of pharmaceutical residues in the overall toxicity of surface water samples, and remark the relevance of monitoring these compounds, as an input for the implementation of future mitigation actions.
... However, caution on the use of hsp70 as a sole biomarker in stress studies is recommended because of tissue-specific upregulation and variation in timing of upregulation after stress exposure (Buckley et al. 2006, Fabbri et al. 2008, Morris et al. 2013). Gill tissue has been shown to have larger and more rapid upregulation compared to other tissues (Buckley et al. 2006, del Rey et al. 2011, Morris et al. 2013, Aleng et al. 2015. Thus, tissue homogenates of very small organisms, like the juvenile P. canaliculus used in this study, may mask significant hsp70 tissue-specific upregulation (Morris et al. 2013). ...
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The marine environment is ever-changing, with daily and seasonal variations in factors such as food availability and seawater temperature. This study assessed the effects of food limitation (i.e. fasting) on heat-stress responses in juveniles (~1.3 mm in shell length) of the green-lipped mussel, Perna canaliculus. Fasting for up to 24 h did not have a significant effect on oxidative damage (protein carbonyls and lipid hydroperoxide accumulation) or the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) after exposure to heat stress. However, fasting for 54 h and subsequent heat stress resulted in increased oxidative damage and decreased activity of antioxidant enzymes in juvenile mussels. On the other hand, gene expression of 70 kDa heat shock protein (hsp70) was only significantly affected by heat shock, rather than nutritional status. Tissue carbohydrate and protein levels were significantly depleted by 54 h of fasting; as these proximate components represent key energy substrates for bivalves, it is suggested that energy limitation contributes to compromised antioxidant activity and predisposition to oxidative damage.
... Caffeine usually causes harmful effects in different marine species in slightly higher concentrations. For example, Del Rey et al. (2011) showed that caffeine concentrations of 0.2 μg/L may have an effect in the gill tissue of the mussel Mytilus californianus at a molecular level (positive regulation of Hsp70). Studies have also shown that the crab Carcinus maenas Ruditapes philippinarum, after the exposure of this mollusk to caffeine for 28 days (0.5, 3.0, and 18.0 μg/L), it was observed that as the concentrations increased, the mollusk lost the ability to prevent lipid peroxidation of cells and also to combat oxidative stress (Cruz et al. 2016). ...
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The aim of this study was to screen and quantify 23 pharmaceutical compounds (including illicit drugs), at two sampling points near the diffusers of the Guarujá submarine outfall, State of São Paulo, Brazil. Samples were collected in triplicate during the high (January 2018) and low (April 2018) seasons at two different water column depths (surface and bottom). A total of 10 compounds were detected using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Caffeine (42.3–141.0 ng/L), diclofenac (3.6–85.7 ng/L), valsartan (4.7–14.3 ng/L), benzoylecgonine (0.3–1.7 ng/L) and cocaine (0.3–0.6 ng/L) were frequently detected (75% ocurrence). Orphenadrine (0.6–3.0 ng/L) and atenolol (0.1–0.3 ng/L), and acetaminophen (1.2–1.4 ng/L) and losartan (0.7–3.4 ng/L), were detected in 50% and 25% of the samples, respectively. Only one sample (12.5%) detected the presence of carbamazepine (<0.001-0.1 ng/L). Unexpectedly a lower frequency of occurrence and concentration of these compounds occurred during the summer season, suggesting that other factors, such as the oceanographic and hydrodynamic regimes of the study area, besides the population rise, should be take into account. Caffeine presented concentrations above the surface water safety limits (0.01 µg/L). For almost all compounds, the observed concentrations indicate non environmental risk for the aquatic biota, except for caffeine, diclofenac and acetaminophen that showed low to moderate ecological risk for the three trophic levels tested.
... 16,17 Once in the aquatic environment, PPCPs may sorb to organic matter, 18 degrade through physicochemical processes, 19 bioaccumulate and biomagnify within a food web, 20 or be metabolized by the organism itself. 21 In addition to their role as indicators of human activity, PPCPs can also be toxicants, disrupting biological and ecological processes 22,23 with physiological, 24 developmental, 25 population, 18 and ecosystem effects. 26 In particular, antibiotics, such as ampicillin, 27 erythromycin, 28 and tetracycline, 29 have been well documented to introduce antibiotic resistance following exposure. ...
Article
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Pharmaceuticals and personal care products (PPCPs) garner increasing attention globally for both their usefulness as indicators of human waste and their potency as emerging organic toxicants. Three decades of rapid increase in PPCP study combined with an increasing number of PPCPs on the global market have created opportunity (1) to review trends in diversity of compounds, sewage treatment techniques (STTs), and ecosystems investigated as well as (2) to identify knowledge gaps in the literature. We conducted a quantitative evidence synthesis of 6,517 abstracts from primary articles in the environmental PPCP literature by examining relative abundance of specific PPCP classes, STTs, and ecosystem types. Our results demonstrate that non-prescription drugs and antibiotics dominated PPCP abstracts, appearing in 51% and 39% of reviewed abstracts respectively, in comparison to hormones (18%), prescription drugs (18%), fragrances (0.3%), and antioxidants (0.0%), which can all elicit physiological and ecological responses even at low concentrations. References to centralized STTs (e.g., activated sludge - 37%) were more frequent than decentralized STTs (e.g., septic - 2%), despite decentralized STTs being common and frequently high impact sources of sewage pollution worldwide. Freshwater lotic systems (63%) were more prevalent than freshwater lentic (24%) and terrestrial (20%) systems. This discrepancy is notable because the longer residence times of lentic and terrestrial systems may enable PPCPs to concentrate and thus increase risk of biological consequences. These results highlight distinct opportunities to address knowledge gaps in the environmental PPCP literature, including underrepresented compounds (e.g., fragrances), sewage treatment techniques (e.g., septic systems), and ecosystem types (e.g., lakes).
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Caffeine has been identified as emerging contaminant of concern due to its widespread occurrence in the aquatic environment and potential to be biologically active. Recently, these concerns have been translated in an increasing research on its occurrence and effects on biota. However, there is still a limited knowledge on the coastal and marine matrices and the implications of their presence in these ecosystems is not fully known. The present review aims to fill these knowledge gaps, analysing the existing literature regarding the occurrence, effects and potential risks of caffeine residues to coastal ecosystems, contributing to the risk assessment of this psychoactive drug in the aquatic environment. The analysed literature reported caffeine concentrations in the coastal ecosystems, raising high concerns about the potential adverse impacts on the ecological safety and human health. Caffeine has been found in tissues from coastal and marine biota including microalgae, coral reefs, bivalves and fish due to bioaccumulation after chronic, long-term exposures in a contaminated environment. Additionally, caffeine residues had been demonstrated to have adverse impacts on aquatic organisms, at environmentally realistic concentrations, inducing oxidative stress and lipid peroxidation, neurotoxicity, changing energy reserves and metabolic activity, affecting reproduction and development and, in some cases, causing mortality. Considering the increasing adverse impacts of caffeine pollution in the coastal environment, this review highlights the urgent need to minimize the increasing load of caffeine to the aquatic ecosystems; being imperative the implementation of scientific programs and projects to classify effectively the caffeine as a high-priority environmentally hazardous emerging pollutant.
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Pharmaceuticals and personal care products in wastewater discharge can be stressors to estuarine species. We transplanted juvenile Pacific oysters at varying distances within sites near wastewater treatment plant outfalls or oyster aquaculture control sites to assess small scale spatial variation in contaminant uptake and oyster condition. Oysters were transplanted to sites in Coos and Netarts Bays, Oregon and Grays Harbor, Washington, then collected after 9 and 12 months. Two pharmaceuticals (miconazole and virginiamycin M1) were detected in spring samples and four alkylphenols (NP1EO, NP2EO, NP and OP) were detected in summer samples, with more frequent detections at wastewater sites. Contaminant concentrations were similar across site types, indicating that even in sparsely populated coastal areas (<25,000 in the watershed), shellfish are exposed to and uptake wastewater contaminants. Additionally, oyster condition was lower at wastewater sites compared to aquaculture sites, indicating a need to better understand whether contaminant exposure affects oyster condition.
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The present work investigates the adsorption of caffeine (CF) by oxidized carbon derived from Luffa cylindrica (OLC). Batch equilibrium experiments were carried out and the effect of adsorption parameters such as pH, contact time, temperature and ionic strength, have been investigated and discussed in detail. Maximum adsorption efficiency was observed at pH 4.0 and the adsorption of CF onto OLC reached equilibrium after 80 min. The kinetic experimental data followed the pseudo-second-order kinetic model and the equilibrium adsorption data fitted well to both Langmuir and Freundlich isotherm models. The maximum adsorption capacity was estimated to be 59.9 mg/g at pH 4 and 298 K. Increasing ionic strength from 0 to 1 M NaClO4 led to a significant decline of the maximum adsorption capacity up to 50%. Thermodynamic studies revealed that the adsorption of CF onto OLC was spontaneous, exothermic with increasing randomness in the solid/solution system interface. The adsorption of CF on OLC at pH values between 4 and 10.4 seems to occur mainly via electrostatic attraction between the negative OLC surface and cationic form of CF. At pH values lower from 3 and higher from 10.4 other mechanisms such as pi-cation and dipol-dipol interactions may determine the CF adsorption on oxidized carbon surfaces.
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Caffeine is one of the most consumed substances, and it has been largely detected in aquatic ecosystems. We investigated the trends in caffeine consumption over three decades and its relationships with gross domestic product (GDP) and human development index (HDI) to understand global patterns and to identify potential hotspots of contamination. The total caffeine consumption is increasing mainly due to population growth. Moreover, caffeine consumption per capita is also increasing in some countries, such as Brazil, Italy, and Ethiopia. A high positive correlation between caffeine consumption per capita with HDI and GDP was found for coffee-importing countries in Europe, while a high negative correlation was found for coffee-exporting countries in Africa. The literature review showed that the highest caffeine concentrations coincide with countries that present an increasing caffeine consumption per capita. Also, approximately 35% of the caffeine concentrations reported in the literature were above the predicted no-effect concentration in the environment and, again, overlaps with countries with increasing per capita consumption. Despite the high degradation rate, caffeine consumption tends to increase in a near future, which may also increase the overall amount of caffeine that comes into the environment, possibly exceeding the thresholds of several species described as tolerant to the current environmental concentrations. Therefore, it is essential to prevent caffeine from reaching aquatic ecosystems, implementing sewage treatment systems, and improving their efficiency.
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The induction of stress-70 (hsp70) and stress-60 (hsp60, chaperonin 60) in the terrestrial woodlouse, Oniscus asellus, after exposure to various heavy metals and heavy metal combinations was investigated by means of standard Western-blot using monoclonal antibodies. The expression of stress-70 was found to react much more sensitively to metal contamination than that of stress-60. The general course of the stress-70 response to increasing metal concentration was found to be similar for all treatments. Based on the obtained results, this course could be divided into three zones: in the first zone, the expression of stress-70 proteins was only marginally increased by comparatively low metal concentrations; in the second zone, strong induction of stress-70 occurred and the ability of the heat shock system to react to increased metal concentrations reached its climax; and in the third, non-compensation zone, a further increase in heavy metal concentration resulted in a decline of stress-70 levels, which can be interpreted as a result of pathological tissue damage. Combinations of metals were found to increase the stress- 70 level more intensely than related concentrations of singularly applied metals. Based on these studies, a ranking scheme of increasing toxicological impact of a variety of heavy metal combinations was composed
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The exposure of organisms to environmental stressors affects the expression levels of certain “stress proteins” that play an important role in protein homeostasis and stress tolerance. We have used an antibody to monitor this response to elevated temperature in the gill, mantle and muscle tissues of the mussel Mytilus edulis. The antibody detected four isoforms of the highly conserved stress-70 family of proteins, which appear homologous with the hsp70, hsc70, hsp72, and grp78 proteins that are detected by the same antibody in humans. Compared with mantle and adductor muscle tissues, gill tissue showed much the greatest increase in levels of both the 70 and 72 kDa proteins. Levels of the 70 and 72 kDa proteins increased for the first 48 hours of heat stress in the gill, and subsequently decreased between 48 and 72 hours. A 78 kDa protein was present in the gill and mantle tissues, but was absent from the adductor muscle. Alternatively, the 70 kDa protein was more abundant in unstressed adductor muscle than in unstressed gill or mantle tissues. Results are discussed in terms of the proposed cellular locations and function of these proteins, the processes contributing to thermally-induced death, and their implications for our understanding of how temperature affects the physiology, ecology and distribution limits of marine organisms.
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The stress response is a series of coordinated physiological reactions increasing an organism's capacity to maintain homeostasis in the presence of threatening agents. This fundamental process is known to involve hormonal signaling to rapidly modulate key physiological functions in vertebrates, but data are lacking concerning neuroendocrine responses to stress in invertebrates. The present study examined circulating catecholamine (CA) responses to stress in oysters. Mechanical disturbances (consisting of shaking the animals) and temperature or salinity variations were applied to the animals because these three types of stressors are commonly encountered by oysters in aquaculture or in their natural habitat. Results show that both circulating noradrenaline (NA) and dopamine (DA) concentrations increased in response to stress. The catecholaminergic response to acute mechanical stressors was rapid (less than 5 min), transient (a return to basal CA levels was observed after 60-90 min), and reflected both the intensity and duration of the perturbation. In contrast, responses to temperature and salinity variations were long lasting (up to 72 h). CA concentrations varied from 1.61 +/- 0.30 ng NA/ml and 0.41 +/- 0.05 ng DA/ml to maximal values of 22.07 +/- 0.97 ng NA/ml and 2.24 +/- 0.19 ng DA/ml. Such CA concentrations are known to induce physiological responses in bivalves, suggesting that stress-induced NA and DA changes exert a regulatory function in oysters.
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This paper briefly reviews the nomenclature and application of stress proteins as biomarkers, then gives a step by step description of the popular methodologies used to measure stress protein in plant and animal tissues together with a critic of each approach and trouble shooting tips. Stress proteins (previously termed heat shock proteins) are induced by many environmental stresses including exposure to trace metals or organic pollutants, changes in temperature or osmolarity, hypoxia/anoxia, and exposure to ultraviolet radiation. Stress proteins, especially hsp70 and hsp60, have been used as biomarkers in a range of algae, invertebrates, fish, and higher vertebrates. Several suggestions are made to improve the utility of stress proteins as a biomarker of exposure e.g., consideration of the kinetics of stress protein induction relative to the pharmacokinetics of pollutant accumulation in the organism of concern, and selection of the type of stress protein for biomonitoring. The methodologies described include sample preparation, western blotting, slot blotting, and antibody detection methods, especially for hsp70. Western blotting has been the most widely applied technique for assaying stress proteins but dot/slot blotting with the inclusion of an internal standard in each gel improves the approach by simplifying the technique—saving time and materials and producing a more quantitative result.
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1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O(2) consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O(2) consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O(2) uptake had been depressed to 50 per cent or more of the normal controls. 6. O(2) consumption-time relationship data indicate an average depression, in O(2) consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O(2) consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O(2) uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.